Development of an Aryloxazole Class of Hepatitis C Virus Inhibitors

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Development of an Aryloxazole Class of Hepatitis C Virus Inhibitors Targeting the Entry Stage of the Viral Replication Cycle Shanshan He,† Kelin Li,‡,⊥ Billy Lin,† Zongyi Hu,† Jingbo Xiao,§ Xin Hu,§ Amy Q. Wang,§ Xin Xu,§ Marc Ferrer,§ Noel Southall,§ Wei Zheng,§ Jeffrey Aubé,‡,⊥ Frank J. Schoenen,‡ Juan J. Marugan,§ T. Jake Liang,*,† and Kevin J. Frankowski*,‡,⊥ †

Liver Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 10 Center Drive, Bethesda, Maryland 20892-1800, United States ‡ University of Kansas Specialized Chemistry Center, University of Kansas, Lawrence, Kansas 66047, United States § Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Drive, Rockville, Maryland 20850, United States S Supporting Information *

ABSTRACT: Reliance on hepatitis C virus (HCV) replicon systems and protein-based screening assays has led to treatments that target HCV viral replication proteins. The model does not encompass other viral replication cycle steps such as entry, processing, assembly and secretion, or viral host factors. We previously applied a phenotypic high-throughput screening platform based on an infectious HCV system and discovered an aryloxazole-based anti-HCV hit. Structure− activity relationship studies revealed several compounds exhibiting EC50 values below 100 nM. Lead compounds showed inhibition of the HCV pseudoparticle entry, suggesting a different mode of action from existing HCV drugs. Hit 7a and lead 7ii both showed synergistic effects in combination with existing HCV drugs. In vivo pharmacokinetics studies of 7ii showed high liver distribution and long half-life without obvious hepatotoxicity. The lead compounds are promising as preclinical candidates for the treatment of HCV infection and as molecular probes to study HCV pathogenesis.



INTRODUCTION Hepatitis C virus (HCV) leads to chronic infection in 80% of patients, and disease progression can eventually cause liver cancer or cirrhosis following decades of asymptomatic infection.1 HCV is the major underlying cause for liver transplants and is responsible for significant healthcare costs.2 The prevalence of HCV has been estimated at around 200 million people worldwide3 and, to date, no effective vaccine has been developed.4 The historical treatment regimen for HCV involved combination therapy of ribavirin with pegylated interferon, a poorly tolerated course of treatment with only moderate success in achieving a six-month post-treatment sustained virological response (SVR), an undetectable viral RNA level six months after treatment cessation.5 The recent approval of several small-molecule direct-acting antivirals (DAAs) has dramatically improved the standard of care for HCV.5 These drugs target the viral proteins (NS3/4A protease, NS5B polymerase, and NS5A) involved in the replication stage of HCV infection. Although these treatments offer renewed hope toward curing HCV infection, the price of the medicines is prohibitively expensive for many high-risk populations such as intravenous drug users, prisoners, and those in the developing world.6 Furthermore, these agents can lead to © 2017 American Chemical Society

rapid development of viral resistance if the virus is not fully eradicated during the treatment regimen. The hegemony of replication inhibitors as treatments for HCV infection is a direct consequence of the available methods to screen for inhibitors of the virus. Current HCV inhibitors have mostly been discovered through either the replicon assay, which measures the replication of isolated viral RNA, or protein-based assays using HCV proteins involved in viral replication (e.g., NS3, NS5A, or NS5B). On the other hand, a cell-based infectious HCV assay platform can cover the complete spectrum of potentially druggable targets in all stages of the HCV replication cycle and allows for the development of inhibitors acting on different phases of the viral life cycle less prone to mutation. More importantly, targeting several key processes in the viral replication cycle may not only increase antiviral efficacy but also reduce the capacity of the virus to develop resistance to the compound. We developed such an assay platform based on an HCV infectious cell culture system.7 The assay was adapted for the high-throughput screening of a 350 000-member compound collection, affording 149 validated Received: May 8, 2017 Published: June 21, 2017 6364

DOI: 10.1021/acs.jmedchem.7b00561 J. Med. Chem. 2017, 60, 6364−6383

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Scheme 1. General Synthetic Route to Aryloxazole Analogues 7a

Reagents and conditions: (a) HCl(g) (4 M in dioxane), AcOH, 0 °C to rt; (b) POCl3, DCE, reflux; (c) KOH, EtOH, isonipecotic acid, rt; (d) HOBt hydrate, DMAP, diisopropylcarbodiimide, DCM, rt or HOBt hydrate, diisopropylcarbodiimide, MeCN, μW irradiation, 100 °C, 10 min. a

Scheme 2. Synthetic Route to Thiazole Analogue 13a

a Reagents and conditions: (a) iPrOH, μW irradiation, 120 °C, 1 h, 65% yield; (b) NCS, MeCN, 60 °C, 2 h, 82% yield; (c) KOH, EtOH, isonipecotic acid, rt, 78% yield; (d) HOBt hydrate, diisopropylcarbodiimide, MeCN, μW irradiation, 100 °C, 10 min, 45% yield.

hit compounds.8 Herein, we describe the optimization, preliminary mode of action (MOA) studies, and detailed biopharmaceutical and pharmacokinetic (PK) properties for an aryloxazole class of compounds discovered during the screening campaign and demonstrate that the class is promising for further pharmaceutical development.

were coupled with the diamine fragments 6 using diisopropylcarbodiimide and 1-hydroxybenzotriazole (HOBt) hydrate to afford the final analogues 7. The coupling reactions were conducted either at room temperature in DCM or at 100 °C using microwave irradiation in MeCN, depending on when during the SAR campaign the analogues were synthesized. Detailed synthetic protocols for individual final analogues are provided in the Experimental Section. The final compounds were purified by either flash chromatography or reverse-phase, preparative-scale, mass-directed high performance liquid chromatography (HPLC), and their purity was assessed by analytical-scale HPLC under analogous conditions. The thiazole core 10 was synthesized via condensation of 3chlorobuta-2-one and thioamide 8 (Scheme 2). Chlorination with N-chlorosuccinimide followed by derivatization with



RESULTS AND DISCUSSION Chemistry. The resynthesized aryloxazole HTS hit compound and most SAR analogues were constructed according to the general route in Scheme 1. The aryloxazole cores 4 were assembled using the method of Goto et al. from commercial benzaldehydes 1 and butane-2,3-dione monooxime 2 via the N-oxide intermediates 3.9 Derivatization of 4 with isonipecotic acid provided the carboxylic acids 5, which 6365

DOI: 10.1021/acs.jmedchem.7b00561 J. Med. Chem. 2017, 60, 6364−6383

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Scheme 3. Representative Synthetic Route to Desmethyloxazole Analogues; Synthesis of 18aa

Reagents and conditions: (a) toluene, 140 °C, 5 h, 57% yield; (b) KOH, EtOH, isonipecotic acid, rt, 93% yield; (d) HOBt hydrate, DMAP, diisopropylcarbodiimide, DCM, rt, 60% yield.

a

isonipecotic acid and diamine fragment 6a as described above afforded the final analogue 13. The desmethyl oxazole core was constructed via condensation and monodehalogenation of 1,3dichloroacetone with benzamide 14 to directly afford the choromethyloxazole scaffold 16 (Scheme 3). Derivatization with isonipecotic acid and diamine fragments 6 as described above afforded the final analogues 18. The 3-piperidinylpropylamines 6 that were not commercially available were readily synthesized by a short two-step sequence (Scheme 4). Briefly, addition of acrylonitrile to the appropriately substituted piperidine followed by Raney nickel-mediated nitrile reduction under a hydrogen atmosphere afforded the requisite diamine fragments 6. Full synthetic protocols for individual diamine fragments are provided in the Supporting Information. Structure−Activity Relationship (SAR) Studies. Shown in Figure 1, the structural elements that were investigated

Scheme 4. General Synthetic Route to Diamine Fragments 6a

a

Reagents and conditions: (a) acrylonitrile, formamide, water; (b) Raney nickel, H2(g), 200 psi, MeOH, NH3(MeOH).

mixture. Both isomers were equipotent in the HCV-Luc assay and showed favorable aqueous solubility; however, 7a possessed slightly lower cytotoxicity and appeared to have better stability in the rat liver microsomes. Replacing the 3,5dimethyl substitution with bulkier 3,5-diethyl (7c) or 3,3,5,5tetramethyl substitution (7d) also afforded potent analogues, though with slightly greater cytotoxicity. Moreover, analogues exploring substitution on the piperidine moiety revealed potency to be dependent on both the size and location of substituents on the heterocycle ring. Sufficiently bulky substitution at the 4-position afforded potent analogues (7e and 7f), while a single methyl group substituent at this position (7g) afforded less potent analogues. Consistent with this trend, the unsubstituted piperidine 7h was even less potent. While the cis-2,6-dimethyl substituted analogue 7i did not improve the potency, the 3-substituted analogues (racemic compounds 7j− 7l) possessed slightly improved potency compared to that of the 3,5-disubstituted compound 7a. The cis-disubstituted compounds (e.g., 7a), however, do not possess any asymmetric centers (meso compounds) and were therefore screened as single stereoisomers rather than enantiomeric mixtures. The constrained analogue 7m did not provide any improvement in potency or cytotoxicity and added unnecessary structural complexity. In all the above cases, increased steric bulk on the piperidine also correlated with decreased microsomal stability. Decreasing or increasing the tether length by one −CH2 group in the diamine fragment (7n and 7o, respectively) did not improve the potency, and we retained the three-carbon linker for the remaining SAR investigation. Ring-opening of the piperidine to the dialkyl amine 7p afforded a slightly less potent analogue, possibly suggesting that the piperidine constraint might not be required. However, limited exploration of more structurally drastic piperidine replacements was detrimental to potency (7q−7s). The relationship between activity and aryl ring substitution is summarized in Table 2. Changing the position of the 2-methyl substituent on the aryl ring to either the 3- or 4-position (7t and 7u, respectively) had no significant effect on potency; however, the 4-methyl analogue 7u had increased cytotoxicity.

Figure 1. Summary of structural modifications explored in optimizing the aryloxazole hit and structure of the selected lead analogue 18a.

during the SAR study are highlighted in the HTS hit molecule (PubChem CID3244725). The majority of the structural analogues focused on varying either aryl-ring substitution or substitution on, or replacement of, the piperidine moiety (the molecular fragments on the left and right ends of the compound hit, respectively). All analogues were screened for inhibition of HCV infection in the cell-based HCV-Luc assay and counterscreened for cytotoxicity in Huh 7.5.1 cells (ATPlite assay). In addition, most analogues were profiled in three assays to assess in vitro biopharmaceutical properties: rat microsomal stability, cell permeability, and aqueous kinetic solubility. Tables 1−3 summarize the results of these SAR investigations. The stereochemistry on the dimethyl-substituted piperidine ring of the HTS hit (PubChem CID3244725) was undefined in the registered structure. Thus, we began our SAR investigation by synthesizing both the cis and trans stereoisomers of the hit compound, 7a and 7b, respectively. The trans stereoisomer 7b was synthesized and screened as a racemic 6366

DOI: 10.1021/acs.jmedchem.7b00561 J. Med. Chem. 2017, 60, 6364−6383

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Table 1. Effect of Varying the Diamine Fragment on Potency, Cytotoxicity, and in Vitro Biopharmaceutical Properties

a

Average of three separate assays (unless otherwise noted) ± standard deviation. bND, not determined. 6367

DOI: 10.1021/acs.jmedchem.7b00561 J. Med. Chem. 2017, 60, 6364−6383

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Table 2. Effect of Varying the Aryl Substitution on Potency, Cytotoxicity, and in Vitro Biopharmaceutical Properties

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Table 2. continued

a

Average of three separate assays (unless otherwise noted) ± standard deviation. bND, not determined. 6369

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Table 3. Effect of Core Scaffold Modifications on Potency, Cytotoxicity, and in Vitro Biopharmaceutical Properties

a

Average of three separate assays (unless otherwise noted) ± standard deviation. bND, not determined.

Figure 2. Titration curves for anti-HCV activity (EC50, triangle in blue) and cytotoxicity (CC50, circle in red) of selected leads. Cell-culture adapted HCV (HCVcc) harboring a luciferase reporter gene (HCV-Luc, genotype 2a) was used to infect Huh7.5.1 cells in the presence of increasing concentrations of test compound. Viral infection and replication were measured by luciferase signal 48 h after treatment. Cytotoxicity was evaluated in parallel with the ATP-based cell viability assay (ATPlite). The results are mean from three replicates ± SEM. The EC50 and CC50 values were calculated with GraphPad Prism 5.0 software using nonlinear regression. Curves and values for each compound were from single representative experiments.

stability. While either monohalogen or methyl substitution was found to afford analogues with attractive potency (95% with the exception of 7yy (purity = 94.2%). The analytical HPLC system used is a dedicated instrument for assessing compound purity and routinely detects impurities as low as 0.1% that elute within the detection window. Any compounds with a measured purity of 100% were thus conservatively assigned a purity of >99.8%. Any compounds purified by reverse-phase, preparative HPLC utilized the same solvent gradient and column material as the analytical conditions to minimize the possibility of impurities that were not detected in the analytical method. All final compounds were inspected for functional groups known to contribute PAINS liabilities, and none were found. General Procedure A-1: Coupling of Carboxylic Acid Fragment 5 or 12 and Amine Fragment 6. To a mixture of oxazolecarboxylic acid 5 or thiazolecarboxylic acid 12, amine 6 (1.0− 2.0 equiv), and HOBt (1.0 equiv) in MeCN (1.5 to 4 mL, ca. 0.1 M) was added diisopropyl carbodiimide (2.0 equiv). The microwave vial was capped and irradiated at 100 °C for 10 min. After cooling to rt, the solvent was removed, and the residue purified by silica gel chromatography (eluents 0−20% MeOH + 0−2% NH4OH(aq) in CH2Cl2) to afford the coupled product. General Procedure A-2: Coupling of Oxazolecarboxylic Acid 5 and Amine Fragment 6. To a mixture of oxazolecarboxylic acid 5, amine 6 (1.0−2.0 equiv), and HOBt (1.0 equiv) in MeCN (1.5 to 4 mL, ca. 0.1 M) was added diisopropyl carbodiimide (2.0 equiv). The microwave vial was capped and irradiated at 100 °C for 10 min. After cooling to rt, the solvent was removed, and the residue was purified by C-18 functionalized silica chromatography (eluents 5−100% MeCN in 6374

DOI: 10.1021/acs.jmedchem.7b00561 J. Med. Chem. 2017, 60, 6364−6383

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Article

1-((5-Methyl-2-(o-tolyl)oxazol-4-yl)methyl)-N-(3-(cis-3,3,5,5-tetramethylpiperidin-1-yl)propyl)piperidine-4-carboxamide 7d. 1-((5Methyl-2-(o-tolyl)oxazol-4-yl)methyl)piperidine-4-carboxylic acid (25 mg, 0.08 mmol) and 3-(3,3,5,5-tetramethylpiperidin-1-yl)propan-1amine (17 mg, 0.09 mmol) were reacted according to general procedure A-3 to afford the product as a light yellow, viscous oil (23 mg, 0.046 mmol, 58% yield). 1H NMR (400 MHz, CDCl3) δ 1.03 (s, 12H), 1.21 (s, 2H), 1.74−1.81 (m, 2H), 1.90−1.95 (complex, 4H), 2.17−2.27 (m, 3H), 2.41 (s, 3H), 2.50−2.57 (m, 2H), 2.63 (s, 4H), 2.66 (s, 3H), 3.21−3.27 (m, 2H), 3.34 (q, J = 6.2 Hz, 2H), 3.69 (s, 2H), 7.24−7.34 (m, 3H), 7.91−7.94 (m, 1H); 13C NMR (101 MHz, CDCl3, APT pulse sequence) δ d (CH, CH3): 10.4, 21.9, 29.5, 41.0, 125.9, 128.6, 129.7, 131.5; u (C, CH2): 24.9, 27.9, 31.4, 38.2, 50.1, 52.1, 52.8, 56.8, 66.1, 126.4, 129.6, 137.1, 147.3, 160.0, 175.1; IR 1557, 1670, 2957 cm−1; HRMS calcd for C30H47N4O2 [M + H]+ 495.3694; found 495.3692; HPLC purity: >99.8%. N-(3-(4-Isopropylpiperidin-1-yl)propyl)-1-((5-methyl-2-(o-tolyl)oxazol-4-yl)methyl)piperidine-4-carboxamide 7e. 1-((5-Methyl-2(o-tolyl)oxazol-4-yl)methyl)piperidine-4-carboxylic acid (65 mg, 0.21 mmol) and 3-(4-isopropylpiperidin-1-yl)propan-1-amine (46 mg, 0.25 mmol) were reacted according to general procedure A-2 to afford the product as a colorless, viscous oil (55 mg, 0.11 mmol, 55% yield). 1H NMR (400 MHz, CDCl3) δ 0.83 (d, J = 6.8 Hz, 6H), 1.08−0.99 (m, 1H), 1.33−1.15 (m, 2H), 1.40 (dq, J = 13.3, 6.4 Hz, 1H), 1.67−1.61 (m, 4H), 1.91−1.73 (m, 6H), 2.14−1.96 (m, 3H), 2.38 (s, 3H), 2.45− 2.39 (m, 2H), 2.65 (s, 3H), 3.08−2.92 (m, 4H), 3.34−3.30 (m, 2H), 3.47 (s, 2H), 7.33−7.17 (m, 3H), 7.59 (t, J = 4.6 Hz, 1H), 7.92 (dd, J = 8.0, 1.7 Hz, 1H); 13C NMR (101 MHz, CDCl3, APT pulse sequence) δ d (CH, CH3): 10.4, 19.6, 21.8, 32.4, 42.2, 43.4, 125.8, 128.7, 129.4, 131.4; u (C, CH2): 24.7, 29.1, 29.4, 40.2, 53.1, 53.8, 54.5, 58.5, 126.8, 132.3, 137.0, 145.6, 159.7, 174.8; IR 1541, 1641, 2938 cm−1; HRMS (m/z): calcd for C29H45N4O2 [M + H]+ 481.3543; found 481.3537; HPLC purity: >99.8%. N-(3-(4,4-Dimethylpiperidin-1-yl)propyl)-1-((5-methyl-2-(o-tolyl)oxazol-4-yl)methyl)piperidine-4-carboxamide 7f. 1-((5-Methyl-2-(otolyl)oxazol-4-yl)methyl)piperidine-4-carboxylic acid (45 mg, 0.14 mmol) and 3-(4,4-dimethylpiperidin-1-yl)propan-1-amine (24 mg, 0.14 mmol) were reacted according to general procedure A-2 to afford the product as a light yellow, viscous oil (47 mg, 0.10 mmol, 70% yield). 1H NMR (400 MHz, CDCl3) δ 0.90 (s, 6H), 1.23−1.28 (m, 2H), 1.39 (t, J = 5.6 Hz, 4H), 1.61−1.68 (m, 2H), 1.75−1.90 (complex, 4H), 2.00−2.13 (complex, 3H), 2.38 (s, 3H), 2.39−2.46 (m, 2H), 2.45 (t, J = 6.2 Hz, 2H), 2.64 (s, 3H), 3.05 (d, J = 11.6 Hz, 2H), 3.30−3.35 (m, 2H), 3.48 (s, 2H), 7.21−7.31 (m, 3H), 7.56 (br s, 1H), 7.90−7.93 (m, 1H); 13C NMR (101 MHz, CDCl3, APT pulse sequence) δ d (CH, CH3): 10.5, 21.8, 43.4, 125.8, 128.7, 129.4, 131.4; u (C, CH2): 24.7, 28.5, 29.1, 38.9, 40.1, 50.3, 53.1, 53.8, 58.4, 126.9, 132.3, 137.0, 145.6, 159.7, 174.8; HRMS (m/z): calcd for C28H43N4O2 [M + H]+ 467.3381; found 467.3389; HPLC purity: >99.8%. N-(3-(4-Methylpiperidin-1-yl)propyl)-1-((5-methyl-2-(o-tolyl)oxazol-4-yl)methyl)piperidine-4-carboxamide 7g. 1-((5-Methyl-2(o-tolyl)oxazol-4-yl)methyl)piperidine-4-carboxylic acid (51 mg, 0.16 mmol) and 3-(4-methylpiperidin-1-yl)propan-1-amine (25 mg, 0.16 mmol) were reacted according to general procedure A-4 to afford the product as a colorless, viscous oil (16 mg, 0.034 mmol, 21% yield). 1H NMR (400 MHz, CDCl3) δ 0.90 (d, J = 6.4 Hz, 3H), 1.18 (dq, J = 3.2, 12.2 Hz, 2H), 1.31−1.43 (m, 1H), 1.62−1.68 (complex, 4H), 1.72− 1.92 (complex, 6H), 1.99−2.13 (complex, 3H), 2.38 (s, 3H), 2.42 (t, J = 6.2 Hz, 2H), 2.65 (s, 3H), 2.91 (d, J = 11.6 Hz, 2H), 3.04 (d, J = 11.6 Hz, 2H), 3.33 (q, J = 5.5 Hz, 2H), 3.48 (s, 2H), 7.21−7.31 (m, 3H), 7.49 (br s, 1H), 7.90−7.93 (m, 1H); 13C NMR (101 MHz, CDCl3, APT pulse sequence) δ d (CH, CH3): 10.5, 21.8, 22.0, 30.7, 43.4, 125.8, 128.7, 129.4, 131.4; u (C, CH2): 24.8, 29.1, 34.5, 40.0, 53.1, 53.8, 54.1, 58.4, 126.9, 132.3, 137.0, 145.6, 159.8, 174.9; HRMS (m/z): calcd for C27H41N4O2 [M + H]+ 453.3224; found 453.3236; HPLC purity: >99.8%. N-(3-(Piperidin-1-yl)propyl)-1-((5-methyl-2-(o-tolyl)oxazol-4-yl)methyl)piperidine-4-carboxamide 7h. 1-((5-Methyl-2-(o-tolyl)oxazol-4-yl)methyl)piperidine-4-carboxylic acid (51 mg, 0.16 mmol) and 3-(piperidin-1-yl)propan-1-amine (23 mg, 0.16 mmol) were

reacted according to general procedure A-4 to afford the product as a colorless, viscous oil (19 mg, 0.044 mmol, 27% yield). 1H NMR (400 MHz, CDCl3) δ 1.41−1.49 (m, 2H), 1.55−1.68 (complex, 6H), 1.73− 1.88 (complex, 5H), 2.00−2.14 (complex, 3H), 2.39 (s, 3H), 2.39− 2.42 (complex, 5H), 2.65 (s, 3H), 3.04 (d, J = 11.6 Hz, 2H), 3.33 (q, J = 6.0 Hz, 2H), 3.48 (s, 2H), 7.22−7.31 (m, 3H), 7.40 (br s, 1H), 7.90−7.94 (m, 1H); 13C NMR (101 MHz, CDCl3, APT pulse sequence) δ d (CH, CH3): 10.5, 21.8, 43.5, 125.8, 128.7, 129.4, 131.4; u (C, CH2): 24.3, 24.7, 26.1, 29.1, 40.0, 53.1, 53.9, 54.7, 58.8, 126.9, 132.4, 137.0, 138.7, 145.6, 174.9; HRMS (m/z): calcd for C26H39N4O2 [M + H]+ 439.3068; found 439.3083; HPLC purity: >99.8%. N-(3-(cis-2,6-Dimethylpiperidin-1-yl)propyl)-1-((5-methyl-2-(otolyl)oxazol-4-yl)methyl)piperidine-4-carboxamide 7i. 1-((5-Methyl2-(o-tolyl)oxazol-4-yl)methyl)piperidine-4-carboxylic acid (78 mg, 0.25 mmol) and 3-(cis-2,6-dimethylpiperidin-1-yl)propan-1-amine (51 mg, 0.30 mmol) were reacted according to general procedure A2 to afford the product as a white solid (81 mg, 0.17 mmol, 70% yield). 1 H NMR (400 MHz, CDCl3) δ 0.53 (q, J = 12.3 Hz, 1H), 0.86 (d, J = 6.6 Hz, 6H), 1.39 (t, J = 11.0 Hz, 2H), 1.58−1.85 (complex, 9H), 1.97−2.14 (m, 3H), 2.39 (s, 3H), 2.42 (t, J = 6.2 Hz, 2H), 2.65 (s, 3H), 2.85−2.89 (m, 2H), 3.01−3.05 (m, 2H), 3.33 (q, J = 6.1 Hz, 2H), 3.48 (s, 2H), 7.22−7.32 (m, 3H), 7.91−7.94 (m, 1H); 13C NMR (101 MHz, CDCl3, APT pulse sequence) δ d (CH, CH3): 10.4, 19.6, 21.8, 31.4, 43.5, 125.8, 128.7, 129.4, 131.4; u (C, CH2): 24.9, 29.1, 40.0, 42.1, 53.1, 53.9, 58.3, 61.8, 126.9, 132.4, 137.0, 145.5, 159.8, 174.8; HRMS calcd for C28H43N4O2 [M + H]+ 467.3386; found 467.3377; HPLC purity: 98.3%. (±)-N-(3-(3-Methylpiperidin-1-yl)propyl)-1-((5-methyl-2-(o-tolyl)oxazol-4-yl)methyl)piperidine-4-carboxamide 7j. 1-((5-Methyl-2-(otolyl)oxazol-4-yl)methyl)piperidine-4-carboxylic acid (70 mg, 0.22 mmol) and 3-(3-methylpiperidin-1-yl)propan-1-amine (42 mg, 0.67 mmol) were reacted according to general procedure A-2 to afford the product as a white solid (81 mg, 0.18 mmol, 80% yield). 1H NMR (400 MHz, CDCl3) δ 0.89 (d, J = 6.5 Hz, 3H), 1.50−1.88 (complex, 13H), 2.01−2.15 (m, 3H), 2.40 (s, 3H), 2.43 (t, J = 6.2 Hz, 2H), 2.66 (s, 3H), 2.84−2.94 (m, 2H), 3.05 (d, J = 11.6 Hz, 2H), 3.35 (q, J = 6.0 Hz, 2H), 3.50 (s, 2H), 7.23−7.33 (m, 3H), 7.37 (br s, 1H), 7.92−7.95 (m, 1H); 13C NMR (101 MHz, CDCl3, APT pulse sequence) δ d (CH, CH3): 10.5, 19.7, 21.8, 31.3, 43.4, 125.8, 128.7, 129.4, 131.4; u (C, CH2): 24.7, 25.6, 29.1, 32.9, 39.9, 53.1, 53.9, 54.1, 58.4, 62.2, 126.9, 132.4, 137.0, 145.5, 159.7, 174.9; HRMS (m/z): calcd for C27H41N4O2 [M + H]+ 453.3224; found 453.3227; HPLC purity: >99.8%. (±)-N-(3-(3-Isopropylpiperidin-1-yl)propyl)-1-((5-methyl-2-(otolyl)oxazol-4-yl)methyl)piperidine-4-carboxamide 7k. 1-((5-Methyl-2-(o-tolyl)oxazol-4-yl)methyl)piperidine-4-carboxylic acid (25 mg, 0.080 mmol) and 3-(3-isopropylpiperidin-1-yl)propan-1-amine (16 mg, 0.087 mmol) were reacted according to general procedure A-3 to afford the product as a light yellow, viscous oil (33 mg, 0.068 mmol, 85% yield). 1H NMR (400 MHz, CDCl3) δ 0.88 (dd, J = 2.5, 6.8 Hz, 6H), 0.95 (dq, J = 3.7, 11.9 Hz, 1H), 1.24−1.34 (m, 2H), 1.40−1.56 (m, 2H), 1.60−1.86 (complex, 9H), 2.00−2.14 (m, 3H), 2.40 (s, 3H), 2.43 (t, J = 6.2 Hz, 2H), 2.66 (s, 3H), 2.88−2.94 (m, 2H), 3.05 (d, J = 11.6 Hz, 2H), 3.31−3.37 (m, 2H), 3.49 (s, 2H), 7.23−7.32 (m, 3H), 7.41 (br s, 1H), 7.92−7.95 (m, 1H); 13C NMR (101 MHz, CDCl3, APT pulse sequence) δ d (CH, CH3): 10.4, 19.8, 20.2, 21.8, 31.1, 42.8, 43.5, 125.8, 128.7, 129.4, 131.4; u (C, CH2): 24.8, 25.8, 27.7, 29.0, 39.9, 53.1, 53.8, 54.4, 58.4, 58.6, 126.9, 132.3, 137.0, 145.6, 159.7, 174.9; HRMS (m/z): calcd for C29H45N4O2 [M + H]+ 481.3543; found 481.3539; HPLC purity: 99.3%. (±)-N-(3-(3-Isobutylpiperidin-1-yl)propyl)-1-((5-methyl-2-(otolyl)oxazol-4-yl)methyl)piperidine-4-carboxamide 7l. 1-((5-Methyl2-(o-tolyl)oxazol-4-yl)methyl)piperidine-4-carboxylic acid (67 mg, 0.21 mmol) and 3-(3-isobutylpiperidin-1-yl)propan-1-amine (42 mg, 0.21 mmol) were reacted according to general procedure A-4 to afford the product as a colorless, viscous oil (22 mg, 0.044 mmol, 21% yield). 1 H NMR (400 MHz, CDCl3) δ 0.88 (dd, J = 2.5, 6.8 Hz, 6H), 0.95 (dq, J = 3.7, 11.9 Hz, 1H), 1.24−1.34 (m, 2H), 1.40−1.56 (m, 2H), 1.60−1.86 (complex, 9H), 2.00−2.14 (m, 3H), 2.40 (s, 3H), 2.43(t, J = 6.2 Hz, 2H), 2.66 (s, 3H), 2.88−2.94 (m, 2H), 3.05 (d, J = 11.6 Hz, 6375

DOI: 10.1021/acs.jmedchem.7b00561 J. Med. Chem. 2017, 60, 6364−6383

Journal of Medicinal Chemistry

Article

(m/z): calcd for C26H41N4O2 [M + H]+ 441.3230; found 441.3226; HPLC purity: >99.8%. 1-((5-Methyl-2-(o-tolyl)oxazol-4-yl)methyl)-N-(3morpholinopropyl)piperidine-4-carboxamide 7q. 1-((5-Methyl-2-(otolyl)oxazol-4-yl)methyl)piperidine-4-carboxylic acid (47 mg, 0.15 mmol) and 3-morpholinopropan-1-amine (43 mg, 0.30 mmol) were reacted according to general procedure A-4 to afford the product as a viscous, light yellow oil (26 mg, 0.060 mmol, 40% yield). 1H NMR (400 MHz, CDCl3) δ 0.86−0.95 (complex, 6H), 1.64−1.70 (m, 2H), 1.72−1.88 (complex, 4H),1.97−2.14 (m, 3H), 2.39 (s, 3H), 2.42−2.50 (complex, 6H), 2.65 (s, 3H), 3.05 (d, J = 11.6 Hz, 2H), 3.34 (q, J = 5.5 Hz, 2H), 3.48 (s, 2H), 3.71 (t, J = 4.6 Hz, 4H), 6.80 (br s, 1H), 7.22− 7.32 (m, 3H), 7.90−7.94 (m, 1H); 13C NMR (101 MHz, CDCl3, APT pulse sequence) δ d (CH, CH3): 10.4, 21.8, 43.5, 125.8, 128.7, 129.4, 131.4; u (C, CH2): 24.9, 29.1, 39.4, 53.1, 53.78, 53.83, 58.1, 67.0, 126.9, 132.3, 137.0, 145.6, 159.8, 174.9; IR 1547, 1642, 2812, 2940 cm−1; HRMS (m/z): calcd for C25H37N4O3 [M + H]+ 441.2860; found 441.2879; HPLC purity: >99.8%. (1-((5-Methyl-2-(o-tolyl)oxazol-4-yl)methyl)piperidin-4-yl)(4-(1methylpiperidin-4-yl)piperazin-1-yl)methanone 7r. 1-((5-Methyl-2(o-tolyl)oxazol-4-yl)methyl)piperidine-4-carboxylic acid (50 mg, 0.16 mmol) and 1-(1-methylpiperidin-4-yl)piperazine (29 mg, 0.16 mmol) were reacted according to general procedure A-4 to afford the product as an off-white solid (36 mg, 0.075 mmol, 47% yield). 1H NMR (400 MHz, CDCl3) δ 1.57 (dq, J = 3.4, 12.0 Hz, 2H), 1.71 (ABq, ΔδAB = 0.08, J = 12.2 Hz, 2H), 1.85−1.96 (complex, 4H), 2.13 (dt, J = 2.0, 11.6 Hz, 2H), 2.21−2.29 (m, 1H), 2.26 (s, 3H), 2.39 (s, 3H), 2.40− 2.47 (m, 1H), 2.48−2.56 (m, 4H), 2.65 (s, 3H), 2.90 (d, J = 11.8 Hz, 2H), 3.05 (d, J = 11.6 Hz, 2H), 3.45−3.51 (m, 2H), 3.51 (s, 2H), 3.58−3.64 (m, 2H), 7.22−7.32 (m, 3H), 7.91−7.94 (m, 1H); 13C NMR (101 MHz, CDCl3, APT pulse sequence) δ d (CH, CH3): 10.5, 21.9, 38.5, 46.1, 61.6, 125.8, 128.7, 129.4, 131.4; u (C, CH2): 28.1, 28.7, 42.0, 45.8, 49.1, 49.4, 52.9, 53.8, 55.4, 126.9, 132.2, 137.0, 145.6, 159.7, 173.3; IR 1446, 1625, 2807, 2938 cm−1; HRMS (m/z): calcd for C28H42N5O2 [M + H]+ 480.3333; found 480.3349; HPLC purity: >99.8%. (4-(2-(Dimethylamino)ethyl)piperazin-1-yl)(1-((5-methyl-2-(otolyl)oxazol-4-yl)methyl)piperidin-4-yl)methanone 7s. 1-((5-Methyl-2-(o-tolyl)oxazol-4-yl)methyl)piperidine-4-carboxylic acid (55 mg, 0.18 mmol) and N,N-dimethyl-2-(piperazin-1-yl)ethanamine (28 mg, 0.18 mmol) were reacted according to general procedure A-4 to afford the product as an off-white solid (30 mg, 0.067 mmol, 38% yield). 1H NMR (400 MHz, CDCl3) δ 1.64−1.71 (m, 2H), 1.90 (dq, J = 2.2, 11.7 Hz, 2H), 2.13 (dt, J = 2.1, 11.6 Hz, 2H), 2.25 (s, 6H), 2.38 (s, 3H), 2.40−2.50 (complex, 5H), 2.59−2.62 (m, 4H), 2.65 (s, 3H), 3.05 (d, J = 11.6 Hz, 2H), 3.47−3.52 (m, 2H), 3.51 (s, 2H), 3.60−3.65 (m, 2H), 7.22−7.31 (m, 3H), 7.91−7.94 (m, 1H); 13C NMR (101 MHz, CDCl3, APT pulse sequence) δ d (CH, CH3): 10.5, 21.8, 38.5, 41.0, 45.9, 125.8, 128.6, 129.4, 131.4; u (C, CH2): 28.7, 41.5, 45.3, 52.9, 53.3, 53.8, 54.1, 56.6, 56.8, 126.9, 132.2, 137.0, 145.6, 159.7, 173.4; IR 1445, 1622, 2816, 2944 cm−1; HRMS (m/z): calcd for C26H40N5O2 [M + H]+ 454.3177; found 454.3192; HPLC purity: 99.3%. N-(3-(cis-3,5-Dimethylpiperidin-1-yl)propyl)-1-((5-methyl-2-(mtolyl)oxazol-4-yl)methyl)piperidine-4-carboxamide 7t. 1-((5-Methyl-2-(m-tolyl)oxazol-4-yl)methyl)piperidine-4-carboxylic acid (30 mg, 0.095 mmol) and 3-(cis-3,5-dimethylpiperidin-1-yl)propan-1-amine (18 mg, 0.10 mmol) were reacted according to general procedure A3 to afford the product as a white solid (29 mg, 0.062 mmol, 65% yield). 1H NMR (400 MHz, CDCl3) δ 0.54 (q, J = 11.9 Hz, 1H), 0.87 (d, J = 6.5 Hz, 6H), 1.41 (t, J = 11.1 Hz, 2H), 1.57−1.88 (complex, 9H), 1.97−2.11 (m, 3H), 2.38 (s, 3H), 2.40 (s, 3H), 2.42 (t, J = 6.1 Hz, 2H), 2.85−2.89 (m, 2H), 2.96−3.05 (m, 2H), 3.33 (q, J = 6.0 Hz, 2H), 3.45 (s, 2H), 7.20−7.23 (m, 1H), 7.28−7.33 (m, 2H), 7.79 (d, J = 7.7 Hz, 1H), 7.86 (br s, 1H); 13C NMR (101 MHz, CDCl3, APT pulse sequence) δ d (CH, CH3): 10.4, 19.6, 21.3, 31.3, 43.4, 123.1, 126.6, 128.5, 130.6; u (C, CH2): 24.9, 29.0, 39.8, 42.1, 53.1, 53.9, 58.1, 61.7, 127.6, 132.6, 138.3, 145.9, 159.5, 174.8; HRMS calcd for C28H43N4O2 [M + H]+ 467.3381; found 467.3388; HPLC purity: 97.2%.

2H), 3.31−3.37 (m, 2H), 3.49 (s, 2H), 7.23−7.32 (m, 3H), 7.41 (br s, 1H), 7.92−7.95 (m, 1H); 13C NMR (101 MHz, CDCl3, APT pulse sequence) δ d (CH, CH3): 10.4, 19.8, 20.2, 21.8, 31.1, 42.8, 43.5, 125.8, 128.7, 129.4, 131.4; u (C, CH2): 24.8, 25.8, 27.7, 29.0, 39.9, 53.1, 53.8, 54.4, 58.4, 58.6, 126.9, 132.3, 137.0, 145.6, 159.7, 174.9; HRMS (m/z): calcd for C30H47N4O2 [M + H]+ 495.3694; found 495.3695; HPLC purity: >99.8%. N-(3-(3-Azabicyclo[3.3.1]nonan-3-yl)propyl)-1-((5-methyl-2-(otolyl)oxazol-4-yl)methyl)piperidine-4-carboxamide 7m. 1-((5-Methyl-2-(o-tolyl)oxazol-4-yl)methyl)piperidine-4-carboxylic acid (62 mg, 0.20 mmol) and 3-(3-azabicyclo[3.3.1]nonan-3-yl)propan-1-amine (55 mg, 0.30 mmol) were reacted according to general procedure A-4 to afford the product as a colorless, viscous oil (21 mg, 0.044 mmol, 22% yield). 1H NMR (400 MHz, CDCl3) δ 1.46−1.55 (m, 3H), 1.60−1.86 (complex, 11H), 2.02−2.15 (complex, 5H), 2.22 (t, J = 6.4 Hz, 2H), 2.31−2.44 (m, 2H), 2.38 (s, 3H), 2.64 (s, 3H), 2.90 (d, J = 10.7 Hz, 2H), 3.05 (d, J = 11.8 Hz, 2H), 3.31 (q, J = 5.6 Hz, 2H), 3.48 (s, 2H), 6.06 (br s, 1H), 7.22−7.31 (m, 3H), 7.91−7.93 (m, 1H); 13C NMR (101 MHz, CDCl3, APT pulse sequence) δ d (CH, CH3): 10.4, 21.8, 29.5, 43.4, 125.8, 128.7, 129.4, 131.4; u (C, CH2): 22.6, 25.8, 29.0, 31.4, 34.1, 38.4, 53.0, 53.8, 57.6, 60.1, 126.9, 132.2, 137.0, 145.6, 159.8, 175.0; HRMS (m/z): calcd for C29H43N4O2 [M + H]+ 479.3381; found 479.3379; HPLC purity: 99.8%. N-(3-(cis-3,5-Dimethylpiperidin-1-yl)ethyl)-1-((5-methyl-2-(otolyl)oxazol-4-yl)methyl)piperidine-4-carboxamide 7n. 1-((5-Methyl-2-(o-tolyl)oxazol-4-yl)methyl)piperidine-4-carboxylic acid (50 mg, 0.16 mmol) and 3-(cis-3,5-dimethylpiperidin-1-yl)ethan-1-amine (30 mg, 0.19 mmol) were reacted according to general procedure A-4 to afford the product as a white solid (59 mg, 0.13 mmol, 82% yield). 1H NMR (400 MHz, CDCl3) δ 0.53 (q, J = 11.9 Hz, 1H), 0.82−0.87 (m, 1H), 0.85 (d, J = 6.5 Hz, 6H), 1.46 (t, J = 11.0 Hz, 2H), 1.58−1.88 (complex, 6H), 2.06−2.15 (m, 3H), 2.38 (s, 3H), 2.42 (t, J = 5.9 Hz, 2H), 2.65 (s, 3H), 2.72−2.82 (m, 2H), 3.01−3.05 (m, 2H), 3.32 (q, J = 5.5 Hz, 2H), 3.47 (s, 2H), 6.33 (br s, 1H), 7.22−7.32 (m, 3H), 7.91−7.94 (m, 1H); 13C NMR (101 MHz, CDCl3, APT pulse sequence) δ d (CH, CH3): 10.4, 19.5, 21.8, 31.1, 43.3, 125.8, 128.7, 129.4, 131.4; u (C, CH2): 28.9, 35.9, 42.1, 53.0, 53.7, 56.6, 61.2, 126.9, 132.2, 137.0, 145.6, 159.8, 175.0; HRMS calcd for C27H41N4O2 [M + H]+ 453.3224; found 453.3221; HPLC purity: 96.6%. N-(3-(cis-3,5-Dimethylpiperidin-1-yl)butyl)-1-((5-methyl-2-(otolyl)oxazol-4-yl)methyl)piperidine-4-carboxamide 7o. 1-((5-Methyl-2-(o-tolyl)oxazol-4-yl)methyl)piperidine-4-carboxylic acid (51 mg, 0.16 mmol) and 3-(cis-3,5-dimethylpiperidin-1-yl)butan-1-amine (36 mg, 0.20 mmol) were reacted according to general procedure A-4 to afford the product as a white solid (24 mg, 0.050 mmol, 31% yield). 1 H NMR (400 MHz, CDCl3) δ 0.52 (q, J = 11.5 Hz, 1H), 0.85 (d, J = 6.5 Hz, 6H), 1.38 (t, J = 11.1 Hz, 2H), 1.49−1.56 (complex, 4H), 1.60−1.72 (m, 3H), 1.75−1.85 (m, 3H), 2.01−2.12 (complex, 4H), 2.29 (t, J = 6.9 Hz, 2H), 2.38 (s, 3H), 2.65 (s, 3H), 2.78−2.83 (m, 2H), 3.04 (d, J = 11.8 Hz, 2H), 3.21−3.26 (m, 2H), 3.47 (s, 2H), 6.22 (br s, 1H), 7.21−7.32 (m, 3H), 7.89−7.94 (m, 1H); 13C NMR (101 MHz, CDCl3, APT pulse sequence) δ d (CH, CH3): 10.4, 19.7, 21.8, 31.1, 43.5, 125.8, 128.7, 129.5, 131.4; u (C, CH2): 24.5, 27.5, 29.0, 39.1, 42.2, 53.0, 53.7, 58.2, 61.6, 126.8, 132.2, 137.0, 145.7, 159.8, 174.9; HRMS calcd for C29H45N4O2 [M + H]+ 481.3537; found 481.3537; HPLC purity: >99.8%. N-(2-(Dipropylamino)ethyl)-1-((5-methyl-2-(o-tolyl)oxazol-4-yl)methyl)piperidine-4-carboxamide 7p. 1-((5-Methyl-2-(o-tolyl)oxazol-4-yl)methyl)piperidine-4-carboxylic acid (50 mg, 0.16 mmol) and N1,N1-dipropylethane-1,2-diamine (25 mg, 0.18 mmol) were reacted according to general procedure A-3 to afford the product as a viscous, light yellow oil (20 mg, 0.046 mmol, 29% yield). 1H NMR (400 MHz, CDCl3) δ 0.86−0.95 (complex, 6H), 1.44−1.71 (complex, 7H), 1.95 (dq, J = 3.0, 12.4 Hz, 2H), 2.09−2.18 (m, 4H), 2.41 (s, 3H), 2.57−2.63 (m, 2H), 2.67 (s, 3H), 2.77 (q, J = 7.0 Hz, 2H), 3.03−3.10 (m, 2H), 3.02−3.12 (m, 2H), 3.38−3.46 (m, 2H), 3.53 (s, 2H), 7.24− 7.33 (m, 3H), 7.93−7.97 (m, 1H); 13C NMR (101 MHz, CDCl3, APT pulse sequence) δ d (CH, CH3): 10.5, 11.2, 11.7, 21.8, 39.0, 125.8, 128.7, 129.4, 131.4; u (C, CH2): 21.0, 22.9, 23.2, 29.0, 46.2, 47.7, 48.7, 50.0, 51.8, 53.0, 53.9, 126.9, 132.3, 137.0, 145.6, 159.7, 175.4; HRMS 6376

DOI: 10.1021/acs.jmedchem.7b00561 J. Med. Chem. 2017, 60, 6364−6383

Journal of Medicinal Chemistry

Article

N-(3-(cis-3,5-Dimethylpiperidin-1-yl)propyl)-1-((5-methyl-2-(ptolyl)oxazol-4-yl)methyl)piperidine-4-carboxamide 7u. 1-((5-Methyl-2-(p-tolyl)oxazol-4-yl)methyl)piperidine-4-carboxylic acid (28 mg, 0.089 mmol) and 3-(cis-3,5-dimethylpiperidin-1-yl)propan-1-amine (17 mg, 0.098 mmol) were reacted according to general procedure A-3 to afford the product as a white solid (28 mg, 0.059 mmol, 66% yield). 1H NMR (400 MHz, CDCl3) δ 0.54 (q, J = 11.9 Hz, 1H), 0.87 (d, J = 6.5 Hz, 6H), 1.41 (t, J = 11.1 Hz, 2H), 1.57−1.88 (complex, 9H), 1.97−2.11 (m, 3H), 2.38 (s, 3H), 2.39 (s, 3H), 2.41 (t, J = 6.1 Hz, 2H), 2.85−2.90 (m, 2H), 2.97−3.02 (m, 2H), 3.33 (q, J = 6.1 Hz, 2H), 3.44 (s, 2H), 7.23 (d, J = 8.0 Hz, 2H), 7.31 (br s, 1H), 7.90 (d, J = 8.2 Hz, 2H); 13C NMR (101 MHz, CDCl3, APT pulse sequence) δ d (CH, CH3): 10.4, 19.6, 21.5, 31.3, 43.5, 126.0, 129.3; u (C, CH2): 24.9, 29.0, 39.9, 42.1, 53.2, 54.0, 58.2, 61.8, 125.1, 132.5, 139.9, 145.6, 159.6, 174.8; HRMS calcd for C28H43N4O2 [M + H]+ 467.3381; found 467.3389; HPLC purity: 99.0%. N-(3-(cis-3,5-Dimethylpiperidin-1-yl)propyl)-1-((2-(2-ethylphenyl)- 5-methyloxazol-4-yl)methyl)piperidine-4-carboxamide 7v. 1((2-(2-Ethylphenyl)- 5-methyloxazol-4-yl)methyl)piperidine-4-carboxylic acid (31 mg, 0.094 mmol) and 3-(cis-3,5-dimethylpiperidin-1yl)propan-1-amine (18 mg, 0.10 mmol) were reacted according to general procedure A-3 to afford the product as a viscous, colorless oil (22 mg, 0.045 mmol, 48% yield). 1H NMR (400 MHz, CDCl3) δ 0.56 (q, J = 11.9 Hz, 1H), 0.87 (d, J = 6.5 Hz, 6H), 1.22 (t, J = 7.5 Hz, 2H), 1.44 (t, J = 11.0 Hz, 2H), 1.59−1.90 (complex, 9H), 1.97−2.18 (m, 3H), 2.40 (s, 3H), 2.44 (t, J = 6.2 Hz, 2H), 2.87−2.92 (m, 2H), 3.01− 3.14 (complex, 4H), 3.34 (q, J = 6.4 Hz, 2H), 3.50 (s, 2H), 7.21−7.39 (complex, 4H), 7.89 (dd, J = 1.4, 7.8 Hz, 1H); 13C NMR (101 MHz, CDCl3, APT pulse sequence) δ d (CH, CH3): 10.4, 15.5, 19.6, 31.2, 43.4, 125.8, 129.1, 129.7, 129.8; u (C, CH2): 24.8, 27.4, 29.1, 39.8, 42.0, 53.0, 53.7, 58.1, 61.7, 126.4, 132.3, 143.3, 145.5, 159.5, 174.9; HRMS calcd for C29H45N4O2 [M + H]+ 481.3537; found 481.3543; HPLC purity: 98.7%. N-(3-(cis-3,5-Dimethylpiperidin-1-yl)propyl)-1-((2-(2,6-dimethylphenyl)-5-methyloxazol-4-yl)methyl)piperidine-4-carboxamide 7w. 1-((2-(2,6-Dimethylphenyl)-5-methyloxazol-4-yl)methyl)piperidine-4carboxylic acid (52 mg, 0.16 mmol) and 3-(cis-3,5-dimethylpiperidin1-yl)propan-1-amine (27 mg, 0.16 mmol) were reacted according to general procedure A-4 to afford the product as a white solid (74 mg, 0.15 mmol, 97% yield). 1H NMR (400 MHz, CDCl3) δ 0.52 (q, J = 11.6 Hz, 1H), 0.83 (d, J = 6.4 Hz, 6H), 1.42 (t, J = 11.1 Hz, 2H), 1.60−1.84 (complex, 9H), 1.94−2.02 (m, 1H), 2.08 (dt, J = 2.8, 11.5 Hz, 2H), 2.19 (s, 6H), 2.34 (s, 3H), 2.42 (t, J = 6.1 Hz, 2H), 2.83− 2.90 (m, 2H), 2.96−3.02 (m, 2H), 3.30 (q, J = 6.1 Hz, 2H), 3.48 (s, 2H), 7.04 (d, J = 7.6 Hz, 2H), 7.18 (t, J = 7.5 Hz, 1H), 7.31 (br s, 1H); 13 C NMR (101 MHz, CDCl3, APT pulse sequence) δ d (CH, CH3): 10.3, 19.5, 20.2, 31.1, 43.3, 127.4, 129.5; u (C, CH2): 24.7, 29.0, 39.6, 41.9, 52.9, 53.5, 58.0, 61.5, 128.5, 131.3, 138.3, 145.7, 158.7, 174.9; HRMS calcd for C29H45N4O2 [M + H]+ 481.3537; found 481.3532; HPLC purity: 98.1%. N-(3-(cis-3,5-Dimethylpiperidin-1-yl)propyl)-1-((2-(3,4-dimethylphenyl)-5-methyloxazol-4-yl)methyl)piperidine-4-carboxamide 7x. 1-((2-(3,4-Dimethylphenyl)-5-methyloxazol-4-yl)methyl)piperidine-4carboxylic acid (25 mg, 0.076 mmol) and 3-(cis-3,5-dimethylpiperidin1-yl)propan-1-amine (16 mg, 0.091 mmol) were reacted according to general procedure A-4 to afford the product as a white solid (34 mg, 0.071 mmol, 93% yield). 1H NMR (400 MHz, CDCl3) δ 0.54 (q, J = 11.2 Hz, 1H), 0.86 (d, J = 6.6 Hz, 6H), 1.41 (t, J = 11.1 Hz, 2H), 1.56−1.84 (complex, 9H), 1.99−2.08 (m, 3H), 2.29 (s, 3H), 2.30 (s, 3H), 2.37 (s, 3H), 2.41 (t, J = 6.2 Hz, 2H), 2.84−2.90 (m, 2H), 3.00 (d, J = 11.2 Hz, 2H), 3.32 (q, J = 6.1 Hz, 2H), 3.43 (s, 2H), 7.17 (d, J = 7.9 Hz, 1H), 7.71 (dd, J = 1.7, 7.8 Hz, 1H), 7.80 (s, 1H); 13C NMR (101 MHz, CDCl3, APT pulse sequence) δ d (CH, CH3): 10.4, 19.6, 19.7, 19.8, 31.3, 43.4, 123.5, 127.2, 129.9; u (C, CH2): 24.9, 28.9, 39.8, 42.0, 53.1, 53.9, 58.1, 61.7, 125.3, 132.3, 136.9, 138.7, 145.6, 159.8, 174.9; HRMS calcd for C29H45N4O2 [M + H]+ 481.3537; found 481.3532; HPLC purity: 95.4%. N-(3-(cis-3,5-Dimethylpiperidin-1-yl)propyl)-1-((2-(2,4-dimethylphenyl)-5-methyloxazol-4-yl)methyl)piperidine-4-carboxamide 7y. 1-((2-(2,4-Dimethylphenyl)-5-methyloxazol-4-yl)methyl)piperidine-4-

carboxylic acid (25 mg, 0.076 mmol) and 3-(cis-3,5-dimethylpiperidin1-yl)propan-1-amine (14 mg, 0.084 mmol) were reacted according to general procedure A-3 to afford the product as a viscous, light yellow oil (23 mg, 0.047 mmol, 62% yield). 1H NMR (400 MHz, CDCl3) δ 0.53 (q, J = 12.0 Hz, 1H), 0.85 (d, J = 6.5 Hz, 6H), 1.41 (t, J = 11.1 Hz, 2H), 1.59−1.84 (complex, 9H), 1.96−2.11 (m, 3H), 2.32 (s, 3H), 2.36 (s, 3H), 2.41 (t, J = 6.2 Hz, 2H), 2.60 (s, 3H), 2.84−2.91 (m, 2H), 3.02 (d, J = 11.7 Hz, 2H), 3.32 (q, J = 6.5 Hz, 2H), 3.46 (s, 2H), 7.03−7.06 (m, 2H), 7.31 (br s, 1H), 7.80 (d, J = 7.7 Hz, 1H); 13C NMR (101 MHz, CDCl3, APT pulse sequence) δ d (CH, CH3): 10.4, 19.6, 21.3, 21.7, 31.3, 43.5, 126.5, 128.7, 132.2; u (C, CH2): 24.8, 29.0, 39.8, 42.0, 53.1, 53.9, 58.2, 61.7, 124.2, 131.9, 136.8, 139.4, 145.2, 159.9, 174.9; HRMS calcd for C29H45N4O2 [M + H]+ 481.3537; found 481.3534; HPLC purity: 99.2%. N-(3-(cis-3,5-Dimethylpiperidin-1-yl)propyl)-1-((2-(3,5-dimethylphenyl)-5-methyloxazol-4-yl)methyl)piperidine-4-carboxamide 7z. 1-((2-(3,5-Dimethylphenyl)-5-methyloxazol-4-yl)methyl)piperidine-4carboxylic acid (25 mg, 0.076 mmol) and 3-(cis-3,5-dimethylpiperidin1-yl)propan-1-amine (14 mg, 0.084 mmol) were reacted according to general procedure A-3 to afford the product as a viscous, colorless oil (19 mg, 0.040 mmol, 52% yield). 1H NMR (400 MHz, CDCl3) δ 0.54 (q, J = 11.9 Hz, 1H), 0.85 (d, J = 6.5 Hz, 6H), 1.42 (t, J = 11.1 Hz, 2H), 1.60−1.84 (complex, 9H), 1.97−2.08 (m, 3H), 2.34 (s, 6H), 2.37 (s, 3H), 2.42 (t, J = 6.1 Hz, 2H), 2.85−2.90 (m, 2H), 2.99 (d, J = 11.6 Hz, 2H), 3.32 (q, J = 6.3 Hz, 2H), 3.43 (s, 2H), 7.03 (s, 1H), 7.29 (br s, 1H), 7.73 (s, 2H); 13C NMR (101 MHz, CDCl3, APT pulse sequence) δ d (CH, CH3): 10.4, 19.6, 21.2, 31.2, 43.4, 123.8, 131.5; u (C, CH2): 24.8, 29.0, 39.8, 42.0, 53.2, 54.0, 58.1, 61.7, 127.5, 132.5, 138.2, 145.8, 159.7, 174.9; HRMS calcd for C29H45N4O2 [M + H]+ 481.3537; found 481.3545; HPLC purity: 99.8%. N-(3-(cis-3,5-Dimethylpiperidin-1-yl)propyl)-1-((2-(2,5-dimethylphenyl)-5-methyloxazol-4-yl)methyl)piperidine-4-carboxamide 7aa. 1-((2-(2,5-Dimethylphenyl)-5-methyloxazol-4-yl)methyl)piperidine-4-carboxylic acid (25 mg, 0.076 mmol) and 3-(cis-3,5dimethylpiperidin-1-yl)propan-1-amine (14 mg, 0.084 mmol) were reacted according to general procedure A-3 to afford the product as a viscous, light yellow oil (8 mg, 0.017 mmol, 22% yield). 1H NMR (400 MHz, CDCl3) δ 0.56 (q, J = 11.4 Hz, 1H), 0.87 (d, J = 6.5 Hz, 6H), 1.46 (t, J = 11.0 Hz, 2H), 1.64−1.87 (complex, 9H), 1.98−2.13 (m, 3H), 2.34 (s, 3H), 2.37 (s, 3H), 2.46 (t, J = 6.0 Hz, 2H), 2.59 (s, 3H), 2.87−2.94 (m, 2H), 3.03 (d, J = 11.6 Hz, 2H), 3.33 (q, J = 6.3 Hz, 2H), 3.47 (s, 2H), 7.07−7.15 (m, 2H), 7.30 (br s, 1H), 7.75 (s, 1H); 13 C NMR (101 MHz, CDCl3, APT pulse sequence) δ d (CH, CH3): 10.4, 19.3, 20.8, 21.3, 30.3, 43.1, 129.2, 130.3, 131.4; u (C, CH2): 24.4, 28.8, 38.5, 41.2, 52.9, 53.7, 56.8, 60.6, 126.5, 131.9, 133.9, 135.3, 145.7, 160.1, 175.3; HRMS calcd for C29H45N4O2 [M + H]+ 481.3537; found 481.3533; HPLC purity: 98.7%. N-(3-(cis-3,5-Dimethylpiperidin-1-yl)propyl)-1-((2-mesityl-5methyloxazol-4-yl)methyl)piperidine-4-carboxamide 7bb. 1-((2Mesityl-5-methyloxazol-4-yl)methyl)piperidine-4-carboxylic acid (25 mg, 0.073 mmol) and 3-(cis-3,5-dimethylpiperidin-1-yl)propan-1amine (14 mg, 0.080 mmol) were reacted according to general procedure A-3 to afford the product as a viscous, light yellow oil (19 mg, 0.039 mmol, 53% yield). 1H NMR (400 MHz, CDCl3) δ 0.54 (q, J = 11.8 Hz, 1H), 0.86 (d, J = 6.5 Hz, 6H), 1.43 (t, J = 11.1 Hz, 2H), 1.62−1.87 (complex, 9H), 1.97−2.04 (m, 1H), 2.10 (dt, J = 2.6, 11.4 Hz, 2H), 2.19 (s, 6H), 2.28 (s, 3H), 2.35 (s, 3H), 2.44 (t, J = 6.2 Hz, 2H), 2.86−2.93 (m, 2H), 3.01 (d, J = 11.6 Hz, 2H), 3.32 (q, J = 6.4 Hz, 2H), 3.50 (s, 2H), 6.88 (s, 2H), 7.33 (br s, 1H); 13C NMR (101 MHz, CDCl3, APT pulse sequence) δ d (CH, CH3): 10.3, 19.6, 20.2, 21.2, 31.1, 43.4, 128.3; u (C, CH2): 24.7, 29.1, 39.7, 42.0, 52.9, 53.6, 58.0, 61.6, 125.7, 131.3, 138.2, 139.3, 145.7, 158.9, 174.9; HRMS calcd for C30H47N4O2 [M + H]+ 495.3694; found 495.3666; HPLC purity: 96.2%. N-(3-(cis-3,5-Dimethylpiperidin-1-yl)propyl)-1-((2-(2,3,4,5,6-pentamethylphenyl)-5-methyloxazol-4-yl)methyl)piperidine-4-carboxamide 7cc. 1-((2-(2,3,4,5,6-Pentamethylphenyl)-5-methyloxazol-4-yl)methyl)piperidine-4-carboxylic acid (25 mg, 0.067 mmol) and 3-(cis3,5-dimethylpiperidin-1-yl)propan-1-amine (13 mg, 0.074 mmol) were reacted according to general procedure A-3 to afford the product as a 6377

DOI: 10.1021/acs.jmedchem.7b00561 J. Med. Chem. 2017, 60, 6364−6383

Journal of Medicinal Chemistry

Article

viscous, light yellow oil (24 mg, 0.045 mmol, 67% yield). 1H NMR (400 MHz, CDCl3) δ 0.54 (q, J = 12.0 Hz, 1H), 0.85 (d, J = 6.5 Hz, 6H), 1.41 (t, J = 11.0 Hz, 2H), 1.58−1.87 (complex, 9H), 1.94−2.02 (m, 1H), 2.01 (s, 6H), 2.10 (dt, J = 2.6, 11.4 Hz, 2H), 2.19 (s, 6H), 2.24 (s, 3H), 2.35 (s, 3H), 2.42 (t, J = 6.1 Hz, 2H), 2.85−2.93 (m, 2H), 3.02 (d, J = 11.6 Hz, 2H), 3.32 (q, J = 5.9 Hz, 2H), 3.52 (s, 2H), 7.33 (br s, 1H); 13C NMR (101 MHz, CDCl3, APT pulse sequence) δ d (CH, CH3): 10.3, 16.3, 16.9, 17.9, 19.6, 31.2, 43.4; u (C, CH2): 24.8, 29.1, 39.9, 42.0, 52.8, 53.5, 58.2, 61.7, 126.9, 130.9, 132.5, 133.5, 136.7, 145.5, 160.4, 174.9; HRMS calcd for C32H51N4O2 [M + H]+ 523.4007; found 523.3984; HPLC purity: >99.8%. N-(3-(cis-3,5-Dimethylpiperidin-1-yl)propyl)-1-((2-(2-chlorophenyl)-5-methyloxazol-4-yl)methyl)piperidine-4-carboxamide 7dd. 1((2-(2-Chlorophenyl)-5-methyloxazol-4-yl)methyl)piperidine-4-carboxylic acid (33 mg, 0.098 mmol) and 3-(cis-3,5-dimethylpiperidin-1yl)propan-1-amine (33 mg, 0.20 mmol) were reacted according to general procedure A-3 to afford the product as a white solid (7 mg, 0.014 mmol, 14% yield). 1H NMR (400 MHz, CDCl3) δ 0.56 (q, J = 12.0 Hz, 1H), 0.89 (d, J = 6.5 Hz, 6H), 1.41 (t, J = 11.0 Hz, 2H), 1.57−1.90 (complex, 9H), 1.98−2.17 (m, 3H), 2.44 (s, 3H), 2.46 (t, J = 6.2 Hz, 2H), 2.86−2.90 (m, 2H), 3.03 (d, J = 11.8 Hz, 2H), 3.34 (q, J = 6.2 Hz, 2H), 3.52 (s, 2H), 7.30−7.40 (m, 2H), 7.45−7.54 (m, 1H), 7.93−8.02 (m, 1H), 8.23 (br s, 1H); 13C NMR (101 MHz, CDCl3, APT pulse sequence) δ d (CH, CH3): 10.5, 19.6, 31.1, 43.4, 126.7, 130.6, 130.9, 131.0; u (C, CH2): 24.8, 29.0, 39.7, 41.9, 53.1, 53.8, 58.0, 61.6, 126.7, 132.2, 132.7, 146.7, 157.3, 174.9; HRMS calcd for C27H40ClN4O2 [M + H]+ 487.2834; found 487.2842; HPLC purity: >99.8%. N-(3-(cis-3,5-Dimethylpiperidin-1-yl)propyl)-1-((2-(3-chlorophenyl)-5-methyloxazol-4-yl)methyl)piperidine-4-carboxamide 7ee. 1((2-(3-Chlorophenyl)-5-methyloxazol-4-yl)methyl)piperidine-4-carboxylic acid (47 mg, 0.14 mmol) and 3-(cis-3,5-dimethylpiperidin-1yl)propan-1-amine (24 mg, 0.14 mmol) were reacted according to general procedure A-4 to afford the product as an off-white solid (52 mg, 0.11 mmol, 76% yield). 1H NMR (400 MHz, CDCl3) δ 0.54 (q, J = 12.4 Hz, 1H), 0.86 (d, J = 6.6 Hz, 6H), 1.40 (t, J = 11.1 Hz, 2H), 1.57−1.87 (complex, 9H), 2.00−2.10 (m, 3H), 2.39 (s, 3H), 2.40 (t, J = 6.2 Hz, 2H), 2.84−2.89 (m, 2H), 2.96−3.02 (m, 2H), 3.32 (q, J = 6.1 Hz, 2H), 3.43 (s, 2H), 7.29 (br s, 1H), 7.35−7.38 (m, 2H), 7.87− 7.90 (m, 1H), 8.00−8.01 (m, 1H); 13C NMR (101 MHz, CDCl3, APT pulse sequence) δ d (CH, CH3): 10.4, 19.6, 31.3, 43.4, 124.1, 126.1, 129.8, 130.0; u (C, CH2): 24.9, 28.9, 39.8, 42.1, 53.1, 53.8, 58.0, 61.7, 129.3, 133.0, 134.7, 146.7, 158.1, 174.9; HRMS calcd for C27H40ClN4O2 [M + H]+ 487.2834; found 487.2829; HPLC purity: >99.8%. N-(3-(cis-3,5-Dimethylpiperidin-1-yl)propyl)-1-((2-(4-chlorophenyl)-5-methyloxazol-4-yl)methyl)piperidine-4-carboxamide 7ff. 1((2-(4-Chlorophenyl)-5-methyloxazol-4-yl)methyl)piperidine-4-carboxylic acid (28 mg, 0.083 mmol) and 3-(cis-3,5-dimethylpiperidin-1yl)propan-1-amine (28 mg, 0.17 mmol) were reacted according to general procedure A-3 to afford the product as a white solid (27 mg, 0.055 mmol, 66% yield). 1H NMR (400 MHz, CDCl3) δ 0.55 (q, J = 11.9 Hz, 1H), 0.87 (d, J = 6.6 Hz, 6H), 1.41 (t, J = 11.1 Hz, 2H), 1.56−1.88 (complex, 9H), 1.95−2.11 (m, 3H), 2.39 (s, 3H), 2.41 (t, J = 6.1 Hz, 2H), 2.85−2.89 (m, 2H), 3.00 (d, J = 11.5 Hz, 2H), 3.33 (q, J = 6.1 Hz, 2H), 3.44 (s, 2H), 7.31 (br s, 1H), 7.38−7.42 (m, 2H), 7.92−7.97 (m, 2H); 13C NMR (101 MHz, CDCl3, APT pulse sequence) δ d (CH, CH3): 10.4, 19.6, 31.4, 43.4, 127.3, 128.9; u (C, CH2): 24.9, 28.9, 39.9, 42.1, 53.2, 53.9, 58.2, 61.8, 126.2, 133.0, 135.8, 146.3, 158.5, 174.8; HRMS calcd for C27H40ClN4O2 [M + H]+ 487.2834; found 487.2841; HPLC purity: >99.8%. N-(3-(cis-3,5-Dimethylpiperidin-1-yl)propyl)-1-((2-(4-fluorophenyl)-5-methyloxazol-4-yl)methyl)piperidine-4-carboxamide 7gg. 1((2-(4-Fluorophenyl)-5-methyloxazol-4-yl)methyl)piperidine-4-carboxylic acid (25 mg, 0.079 mmol) and 3-(cis-3,5-dimethylpiperidin-1yl)propan-1-amine (15 mg, 0.086 mmol) were reacted according to general procedure A-3 to afford the product as a white solid (25 mg, 0.054 mmol, 69% yield). 1H NMR (400 MHz, CDCl3) δ 0.55 (q, J = 11.9 Hz, 1H), 0.87 (d, J = 6.6 Hz, 6H), 1.41 (t, J = 11.1 Hz, 2H), 1.56−1.88 (complex, 9H), 1.95−2.11 (m, 3H), 2.39 (s, 3H), 2.41 (t, J

= 6.1 Hz, 2H), 2.85−2.89 (m, 2H), 3.00 (d, J = 11.5 Hz, 2H), 3.33 (q, J = 6.1 Hz, 2H), 3.44 (s, 2H), 7.31 (br s, 1H), 7.38−7.42 (m, 2H), 7.92−7.97 (m, 2H); 13C NMR (101 MHz, CDCl3, APT pulse sequence) δ d (CH, CH3): 10.4, 19.4, 30.8, 43.3, 115.7 (d, J = 22.1 Hz), 128.1 (d, J = 8.5 Hz); u (C, CH2): 24.7, 28.8, 39.2, 41.7, 53.1, 53.9, 57.5, 61.2, 124.1 (d, J = 3.2 Hz), 132.6, 146.1, 158.6 (d, J = 0.4 Hz), 163.7 (d, J = 250.9 Hz), 175.0; HRMS calcd for C27H40FN4O2 [M + H]+ 471.3130; found 471.3114; HPLC purity: >99.8%. N-(3-(cis-3,5-Dimethylpiperidin-1-yl)propyl)-1-((2-(2,6-dichlorophenyl)-5-methyloxazol-4-yl)methyl)piperidine-4-carboxamide 7hh. 1-((2-(2,5-Dichlorophenyl)-5-methyloxazol-4-yl)methyl)piperidine-4-carboxylic acid (25 mg, 0.068 mmol) and 3-(cis-3,5dimethylpiperidin-1-yl)propan-1-amine (13 mg, 0.074 mmol) were reacted according to general procedure A-4 to afford the product as a viscous, light yellow oil (21 mg, 0.041 mmol, 60% yield). 1H NMR (400 MHz, CDCl3) δ 0.56 (q, J = 11.9 Hz, 1H), 0.88 (d, J = 6.5 Hz, 6H), 1.44 (t, J = 11.0 Hz, 2H), 1.64−1.91 (complex, 9H), 1.97−2.06 (m, 1H), 2.12 (dt, J = 2.6, 11.4 Hz, 2H), 2.42 (s, 3H), 2.45 (t, J = 6.1 Hz, 2H), 2.88−2.92 (m, 2H), 3.00−3.03 (m, 2H), 3.34 (q, J = 6.1 Hz, 2H), 3.56 (s, 2H), 7.30−7.43 (complex, 4H); 13C NMR (101 MHz, CDCl3, APT pulse sequence) δ d (CH, CH3): 10.4, 19.6, 31.2, 43.4, 128.0, 131.6; u (C, CH2): 24.7, 29.1, 39.8, 42.0, 52.8, 53.5, 58.1, 61.6, 128.3, 132.0, 136.4, 147.1, 154.0, 174.9; HRMS calcd for C27H39Cl2N4O2 [M + H]+ 521.2445; found 521.2439; HPLC purity: >99.8%. N-(3-(cis-3,5-Dimethylpiperidin-1-yl)propyl)-1-((2-(3,4-dichlorophenyl)-5-methyloxazol-4-yl)methyl)piperidine-4-carboxamide 7ii. 1-((2-(3,4-Dichlorophenyl)-5-methyloxazol-4-yl)methyl)piperidine-4carboxylic acid (25 mg, 0.068 mmol) and 3-(cis-3,5-dimethylpiperidin1-yl)propan-1-amine (13 mg, 0.074 mmol) were reacted according to general procedure A-4 to afford the product as a viscous, light yellow oil (28 mg, 0.053 mmol, 78% yield). 1H NMR (400 MHz, CDCl3) δ 0.71 (q, J = 12.9 Hz, 1H), 0.93 (d, J = 6.1 Hz, 6H), 1.82−2.10 (complex, 11H), 2.15−2.32 (m, 3H), 2.37 (s, 3H), 2.94 (t, J = 7.3 Hz, 2H), 3.05−3.14 (m, 2H), 3.23−3.34 (m, 4H), 3.54 (s, 2H), 7.18 (br s, 1H), 7.48 (d, J = 8.4 Hz, 1H), 7.81 (dd, J = 2.0, 8.4 Hz, 1H), 8.08 (d, J = 2.0 Hz, 1H); 13C NMR (101 MHz, CDCl3, APT pulse sequence) δ d (CH, CH3): 10.4, 18.8, 28.8, 41.0, 125.1, 127.9, 130.8; u (C, CH2): 23.9, 28.0, 28.0, 36.4, 40.0, 52.6, 53.1, 58.9, 127.4, 133.1, 134.0, 157.6, 162.5, 162.8, 175.6; IR 1541, 1641, 2922, 2947 cm−1; HRMS calcd for C27H39Cl2N4O2 [M + H]+ 521.2445; found 521.2437; HPLC purity: 99.5%. N-(3-(cis-3,5-Dimethylpiperidin-1-yl)propyl)-1-((2-(2,6-difluorophenyl)-5-methyloxazol-4-yl)methyl)piperidine-4-carboxamide 7jj. 1-((2-(2,6-Difluorophenyl)-5-methyloxazol-4-yl)methyl)piperidine-4carboxylic acid (25 mg, 0.074 mmol) and 3-(cis-3,5-dimethylpiperidin1-yl)propan-1-amine (14 mg, 0.082 mmol) were reacted according to general procedure A-3 to afford the product as a viscous, light yellow oil (12 mg, 0.024 mmol, 33% yield). 1H NMR (400 MHz, CDCl3) δ 0.56 (q, J = 12.3 Hz, 1H), 0.87 (d, J = 6.5 Hz, 6H), 1.49 (t, J = 10.4 Hz, 2H), 1.67−1.86 (complex, 9H), 1.98−2.12 (m, 3H), 2.40 (s, 3H), 2.48 (t, J = 5.4 Hz, 2H), 2.89−2.96 (m, 2H), 3.00 (d, J = 11.6 Hz, 2H), 3.33 (q, J = 6.4 Hz, 2H), 3.50 (s, 2H), 6.99 (t, J = 8.3 Hz, 2H), 7.29− 7.40 (m, 2H); 13C NMR (101 MHz, CDCl3, APT pulse sequence) δ d (CH, CH3): 10.5, 19.5, 30.9, 43.3, 112.0 (d, J = 25.6 Hz), 131.4 (t, J = 10.5 Hz); u (C, CH2): 24.6, 29.0, 39.3, 41.7, 53.1, 53.8, 57.7, 61.3, 107.0 (d, J = 16.1 Hz), 132.9, 147.2, 150.5 (d, J = 3.1 Hz), 160.7 (dd, J = 5.9, 257.3 Hz), 175.0; HRMS calcd for C27H39F2N4O2 [M + H]+ 489.3036; found 489.3040; HPLC purity: 97.3%. N-(3-(cis-3,5-Diethylpiperidin-1-yl)propyl)-1-((2-(2,6-difluorophenyl)-5-methyloxazol-4-yl)methyl)piperidine-4-carboxamide 7kk. 1-((2-(2,6-Difluorophenyl)-5-methyloxazol-4-yl)methyl)piperidine-4-carboxylic acid (25 mg, 0.074 mmol) and 3-(cis-3,5diethylpiperidin-1-yl)propan-1-amine (16 mg, 0.082 mmol) were reacted according to general procedure A-3 to afford the product as a viscous, light yellow oil (19 mg, 0.036 mmol, 49% yield). 1H NMR (400 MHz, CDCl3) δ 0.49 (q, J = 12.4 Hz, 1H), 0.91 (d, J = 7.5 Hz, 6H), 1.19−1.29 (m, 4H), 1.14−1.49 (m, 4H), 1.66−1.89 (complex, 7H), 2.00−2.13 (m, 3H), 2.43 (s, 3H), 2.45 (t, J = 6.0 Hz, 2H), 2.94− 2.98 (m, 2H), 2.99−3.06 (m, 2H), 3.35 (q, J = 6.3 Hz, 2H), 3.53 (s, 6378

DOI: 10.1021/acs.jmedchem.7b00561 J. Med. Chem. 2017, 60, 6364−6383

Journal of Medicinal Chemistry

Article

2H), 7.02 (t, J = 8.4 Hz, 2H), 7.34−7.43 (m, 2H); 13C NMR (101 MHz, CDCl3, APT pulse sequence) δ d (CH, CH3): 10.5, 11.4, 38.0, 43.5, 112.0 (d, J = 25.6 Hz), 131.4 (t, J = 10.5 Hz); u (C, CH2): 24.9, 27.4, 29.0, 37.3, 39.9, 53.1, 53.8, 58.4, 60.4, 106.8 (d, J = 16.3 Hz), 132.9, 147.1, 150.5, 160.7 (d, J = 257.3 Hz), 174.9; HRMS calcd for C29H43F2N4O2 [M + H]+ 517.3349; found 517.3351; HPLC purity: >99.8%. N-(3-(cis-3,5-Dimethylpiperidin-1-yl)propyl)-1-((2-(2,6-dibromophenyl)-5-methyloxazol-4-yl)methyl)piperidine-4-carboxamide 7ll. 1-((2-(2,5-Dibromophenyl)-5-methyloxazol-4-yl)methyl)piperidine-4carboxylic acid (25 mg, 0.055 mmol) and 3-(cis-3,5-dimethylpiperidin1-yl)propan-1-amine (10 mg, 0.060 mmol) were reacted according to general procedure A-3 to afford the product as a viscous, light yellow oil (22 mg, 0.037 mmol, 67% yield). 1H NMR (400 MHz, CDCl3) δ 0.56 (q, J = 12.2 Hz, 1H), 0.88 (d, J = 6.5 Hz, 6H), 1.43 (t, J = 11.0 Hz, 2H), 1.61−1.90 (complex, 9H), 1.98−2.02 (m, 1H), 2.14 (dt, J = 2.8, 11.5 Hz, 2H), 2.42 (s, 3H), 2.44 (t, J = 6.0 Hz, 2H), 2.87−2.93 (m, 2H), 2.99−3.05 (m, 2H), 3.34 (q, J = 6.3 Hz, 2H), 3.58 (s, 2H), 7.20 (t, J = 8.1 Hz, 1H), 7.37 (br s, 1H), 7.62 (d, J = 8.1 Hz, 1H); 13C NMR (101 MHz, CDCl3, APT pulse sequence) δ d (CH, CH3): 10.4, 19.6, 31.3, 43.4, 131.6, 132.3; u (C, CH2): 24.7, 29.1, 39.9, 42.0, 52.6, 53.3, 58.2, 61.7, 125.3, 131.7, 132.2, 146.8, 156.4, 174.9; HRMS calcd for C27H39Br2N4O2 [M + H]+ 609.1434; found 609.1430; HPLC purity: >99.8%. N-(3-(cis-3,5-Dimethylpiperidin-1-yl)propyl)-1-((2-(2-bromo-6chlorophenyl)-5-methyloxazol-4-yl)methyl)piperidine-4-carboxamide 7mm. 1-((2-(2-Bromo-6-chlorophenyl)-5-methyloxazol-4-yl)methyl)piperidine-4-carboxylic acid (25 mg, 0.060 mmol) and 3-(cis3,5-dimethylpiperidin-1-yl)propan-1-amine (11 mg, 0.066 mmol) were reacted according to general procedure A-3 to afford the product as a white solid (23 mg, 0.041 mmol, 68% yield). 1H NMR (400 MHz, CDCl3) δ 0.56 (q, J = 11.8 Hz, 1H), 0.88 (d, J = 6.5 Hz, 6H), 1.44 (t, J = 11.0 Hz, 2H), 1.61−1.90 (complex, 9H), 1.98−2.04 (m, 1H), 2.13 (dt, J = 2.8, 11.5 Hz, 2H), 2.42 (s, 3H), 2.45 (t, J = 6.1 Hz, 2H), 2.86− 2.95 (m, 2H), 2.98−3.05 (m, 2H), 3.34 (q, J = 6.3 Hz, 2H), 3.57 (s, 2H), 7.27 (t, J = 8.1 Hz, 1H), 7.36 (br s, 1H), 7.44 (dd, J = 1.1, 8.1 Hz, 1H), 7.58 (dd, J = 1.1, 8.1 Hz, 1H); 13C NMR (101 MHz, CDCl3, APT pulse sequence) δ d (CH, CH3): 10.4, 19.6, 31.2, 43.4, 128.5, 131.1, 131.9; u (C, CH2): 24.7, 29.1, 39.8, 42.0, 52.7, 53.4, 58.2, 61.7, 125.4, 130.3, 131.8, 136.4, 147.0, 155.2, 174.9; HRMS calcd for C27H39BrClN4O2 [M + H]+ 565.1939; found 565.1939; HPLC purity: >99.8%. N-(3-(cis-3,5-Dimethylpiperidin-1-yl)propyl)-1-((2-(2-chloro-6methylphenyl)-5-methyloxazol-4-yl)methyl)piperidine-4-carboxamide 7nn. 1-((2-(2-Chloro-6-methylphenyl)-5-methyloxazol-4-yl)methyl)piperidine-4-carboxylic acid (80 mg, 0.23 mmol) and 3-(cis3,5-dimethylpiperidin-1-yl)propan-1-amine (59 mg, 0.078 mmol) were reacted according to general procedure A-4 to afford the product as a white solid (71 mg, 0.14 mmol, 62% yield). 1H NMR (400 MHz, CDCl3) δ 0.53 (q, J = 12.0 Hz, 1H), 0.86 (d, J = 6.5 Hz, 6H), 1.39 (t, J = 11.0 Hz, 2H), 1.53−1.89 (complex, 9H), 1.96−2.04 (m, 1H), 2.11 (dt, J = 2.8, 11.5 Hz, 2H), 2.24 (s, 3H), 2.39 (s, 3H), 2.40 (t, J = 6.0 Hz, 2H), 2.84−2.88 (m, 2H), 2.99−3.04 (m, 2H), 3.32 (q, J = 6.0 Hz, 2H), 3.53 (s, 2H), 7.14−7.17 (m, 1H), 7.23−7.31 (m, 2H), 7.36 (br s, 1H); 13C NMR (101 MHz, CDCl3, APT pulse sequence) δ d (CH, CH3): 10.3, 19.6, 20.2, 31.4, 43.4, 126.9, 128.3, 130.7; u (C, CH2): 24.8, 29.1, 40.0, 42.1, 52.8, 53.5, 58.3, 61.8, 128.3, 131.7, 134.8, 140.9, 146.5, 156.2, 174.8; HRMS calcd for C28H42ClN4O2 [M + H]+ 501.2991; found 501.2995; HPLC purity: 96.1%. (±)-1-((2-(2-Chloro-6-methylphenyl)-5-methyloxazol-4-yl)methyl)-N-(3-(3-methylpiperidin-1-yl)propyl)piperidine-4-carboxamide 7oo. 1-((2-(2-Chloro-6-methylphenyl)-5-methyloxazol-4-yl)methyl)piperidine-4-carboxylic acid (25 mg, 0.072 mmol) and 3-(3methylpiperidin-1-yl)propan-1-amine (12 mg, 0.079 mmol) were reacted according to general procedure A-3 to afford the product as a viscous, light yellow oil (20 mg, 0.041 mmol, 56% yield). 1H NMR (400 MHz, CDCl3) δ 0.80−0.91 (m, 1H), 0.85 (d, J = 6.4 Hz, 3H), 1.46−1.88 (complex, 12H), 1.96−2.04 (m, 1H), 2.09 (dt, J = 2.8, 11.5 Hz, 2H), 2.22 (s, 3H), 2.37 (s, 3H), 2.40 (t, J = 6.2 Hz, 2H), 2.81− 2.90 (m, 2H), 2.97−3.03 (m, 2H), 3.30 (q, J = 6.2 Hz, 2H), 3.51 (s,

2H), 7.12−7.15 (m, 1H), 7.22−7.28 (m, 2H), 7.38 (br s, 1H); 13C NMR (101 MHz, CDCl3, APT pulse sequence) δ d (CH, CH3): 10.3, 19.7, 20.2, 31.3, 43.4, 126.9, 128.3, 130.7; u (C, CH2): 24.7, 25.6, 29.0, 32.9, 39.9, 52.8, 53.5, 54.1, 58.4, 62.1, 128.3, 131.6, 134.8, 140.9, 146.5, 156.2, 174.9; HRMS calcd for C27H40ClN4O2 [M + H]+ 487.2834; found 487.2839; HPLC purity: >99.8%. 1-((2-(2-Chloro-6-methylphenyl)-5-methyloxazol-4-yl)methyl)-N(3-(4,4-dimethylpiperidin-1-yl)propyl)piperidine-4-carboxamide 7pp. 1-((2-(2-Chloro-6-methylphenyl)-5-methyloxazol-4-yl)methyl)piperidine-4-carboxylic acid (25 mg, 0.072 mmol) and 3-(4,4dimethylpiperidin-1-yl)propan-1-amine (13 mg, 0.079 mmol) were reacted according to general procedure A-3 to afford the product as a viscous, light yellow oil (20 mg, 0.039 mmol, 55% yield). 1H NMR (400 MHz, CDCl3) 0.92 (s, 6H), 1.40 (t, J = 5.7 Hz, 4H), 1.63−1.69 (m, 2H), 1.73−1.93 (complex, 4H), 1.98−2.07 (m, 1H), 2.12 (dt, J = 2.6, 11.5 Hz, 2H), 2.25 (s, 3H), 2.39 (s, 3H), 2.39−2.44 (m, 4H), 2.46 (t, J = 6.1 Hz, 2H), 3.03 (d, J = 11.7 Hz, 2H), 3.33 (q, J = 5.92 Hz, 2H), 3.53 (s, 2H), 7.12−7.20 (m, 1H), 7.22−7.33 (m, 2H), 7.54 (br s, 1H); 13C NMR (101 MHz, CDCl3, APT pulse sequence) δ d (CH, CH3): 10.3, 20.3, 41.0, 43.4, 126.9, 128.3, 130.7; u (C, CH2): 24.7, 28.5, 29.1, 38.9, 40.1, 50.3, 52.9, 53.5, 58.3, 128.3, 131.6, 134.8, 140.9, 146.5, 156.2, 174.9; HRMS calcd for C28H42ClN4O2 [M + H]+ 501.2991; found 501.2985; HPLC purity: >99.8%. 1-((2-(2-Chloro-6-methylphenyl)-5-methyloxazol-4-yl)methyl)-N(3-(4-isopropylpiperidin-1-yl)propyl)piperidine-4-carboxamide 7qq. 1-((2-(2-Chloro-6-methylphenyl)-5-methyloxazol-4-yl)methyl)piperidine-4-carboxylic acid (25 mg, 0.072 mmol) and 3-(4isopropylpiperidin-1-yl)propan-1-amine (15 mg, 0.079 mmol) were reacted according to general procedure A-3 to afford the product as a viscous, light yellow oil (21 mg, 0.041 mmol, 57% yield). 1H NMR (400 MHz, CDCl3) δ 0.87 (d, J = 6.8 Hz, 6H), 1.02−1.09 (m, 1H), 1.38−1.49 (m, 1H), 1.61−1.71 (m, 4H), 1.73−1.93 (complex, 6H), 1.99−2.05 (m, 1H), 2.11 (dt, J = 2.6, 11.5 Hz, 2H), 2.25 (s, 3H), 2.39 (s, 3H), 2.42 (t, J = 6.0 Hz, 2H), 3.01 (t, J = 10.5 Hz, 4H), 3.34 (q, J = 6.0 Hz, 2H), 3.53 (s, 2H), 7.13−7.20 (m, 1H), 7.23−7.34 (m, 2H), 7.52 (br s, 1H); 13C NMR (101 MHz, CDCl3, APT pulse sequence) δ d (CH, CH3): 10.3, 19.7, 20.3, 32.4, 42.3, 43.4, 126.9, 128.3, 130.7; u (C, CH2): 24.8, 29.1, 29.4, 40.1, 52.9, 53.5, 54.5, 58.4, 128.3, 131.6, 134.8, 140.9, 146.5, 156.2, 174.8; HRMS calcd for C29H44ClN4O2 [M + H]+ 515.3147; found 515.3149; HPLC purity: >99.8%. N-(3-(cis-3,5-Dimethylpiperidin-1-yl)propyl)-1-((2-(2-chloro-6(trifluoromethyl)phenyl)-5-methyloxazol-4-yl)methyl)piperidine-4carboxamide 7rr. 1-((2-(2-Chloro-6-(trifluoromethyl)phenyl)-5methyloxazol-4-yl)methyl)piperidine-4-carboxylic acid (24 mg, 0.061 mmol) and 3-(cis-3,5-dimethylpiperidin-1-yl)propan-1-amine (11 mg, 0.067 mmol) were reacted according to general procedure A-4 to afford the product as a viscous, light yellow oil (20 mg, 0.035 mmol, 58% yield). 1H NMR (400 MHz, CDCl3) δ 0.55 (q, J = 11.9 Hz, 1H), 0.87 (d, J = 6.6 Hz, 6H), 1.41 (t, J = 11.0 Hz, 2H), 1.62−1.91 (complex, 9H), 1.96−2.01 (m, 1H), 2.12 (dt, J = 2.7, 11.5 Hz, 2H), 2.41 (s, 3H), 2.43 (t, J = 5.9 Hz, 2H), 2.85−2.93 (m, 2H), 2.97−3.02 (m, 2H), 3.35 (q, J = 6.4 Hz, 2H), 3.57 (s, 2H), 7.41 (br s, 1H) 7.54− 7.59 (m, 1H), 7.70 (d, J = 8.4 Hz, 2H); 13C NMR (101 MHz, CDCl3, APT pulse sequence) δ d (CH, CH3): 10.3, 19.6, 31.3, 43.4, 124.6 (q, J = 4.9 Hz), 131.3, 132.9; u (C, CH2): 24.7, 29.1, 40.1, 42.1, 52.6, 53.3, 58.4, 61.8, 125.5 (q, J = 286.8 Hz), 131.6, 1325 (q, J = 31.6 Hz), 137.1, 147.7, 153.1, 155.3, 175.4; HRMS calcd for C29H39ClF3N4O2 [M + H]+ 555.2708; found 555.2705; HPLC purity: >99.8%. N-(3-(cis-3,5-Dimethylpiperidin-1-yl)propyl)-1-((2-(2-fluoro-6methylphenyl)-5-methyloxazol-4-yl)methyl)piperidine-4-carboxamide 7ss. 1-((2-(2-Fluoro-6-methylphenyl)-5-methyloxazol-4-yl)methyl)piperidine-4-carboxylic acid (25 mg, 0.075 mmol) and 3-(cis3,5-dimethylpiperidin-1-yl)propan-1-amine (14 mg, 0.083 mmol) were reacted according to general procedure A-4 to afford the product as a viscous, light yellow oil (22 mg, 0.046 mmol, 61% yield). 1H NMR (400 MHz, CDCl3) δ 0.56 (q, J = 12.3 Hz, 1H), 0.88 (d, J = 6.6 Hz, 6H), 1.41 (t, J = 11.0 Hz, 2H), 1.58−1.91 (complex, 9H), 1.96−2.06 (m, 1H), 2.11 (dt, J = 3.0, 11.4 Hz, 2H), 2.41 (s, 3H), 2.43 (s, 3H), 2.43 (t, J = 6.2 Hz, 2H), 2.86−2.91 (m, 2H), 2.99−3.09 (m, 2H), 3.35 (q, J = 6.0 Hz, 2H), 3.52 (s, 2H), 6.99 (t, J = 9.3 Hz, 1H), 7.07 (d, J = 6379

DOI: 10.1021/acs.jmedchem.7b00561 J. Med. Chem. 2017, 60, 6364−6383

Journal of Medicinal Chemistry

Article

7.7 Hz, 1H), 7.27−7.33 (m, 1H), 7.35 (br s, 1H); 13C NMR (101 MHz, CDCl3, APT pulse sequence) δ d (CH, CH3): 10.4, 19.6, 20.4, 31.4, 43.5, 113.2 (d, J = 22.1 Hz), 126.0, 130.8; u (C, CH2): 24.8, 29.1, 40.0, 42.1, 53.0, 53.7, 58.3, 61.8, 116.7 (d, J = 13.9 Hz), 132.2, 140.6 (d, J = 1.5 Hz), 146.4, 154.4, 161.1 (d, J = 252.2 Hz) 174.8; HRMS calcd for C28H42FN4O2 [M + H]+ 485.3286; found 485.3278; HPLC purity: >99.8%. N-(3-(cis-3,5-Dimethylpiperidin-1-yl)propyl)-1-((2-(2-bromo-6methoxyphenyl)-5-methyloxazol-4-yl)methyl)piperidine-4-carboxamide 7tt. 1-((2-(2-Bromo-6-methoxyphenyl)-5-methyloxazol-4-yl)methyl)piperidine-4-carboxylic acid (25 mg, 0.061 mmol) and 3-(cis3,5-dimethylpiperidin-1-yl)propan-1-amine (11 mg, 0.067 mmol) were reacted according to general procedure A-3 to afford the product as a viscous, light yellow oil (26 mg, 0.046 mmol, 76% yield). 1H NMR (400 MHz, CDCl3) δ 0.56 (q, J = 11.9 Hz, 1H), 0.88 (d, J = 6.5 Hz, 6H), 1.45 (t, J = 11.0 Hz, 2H), 1.65−1.90 (complex, 9H), 1.98−2.05 (m, 1H), 2.13 (dt, J = 2.9, 11.5 Hz, 2H), 2.40 (s, 3H), 2.46 (t, J = 6.1 Hz, 2H), 2.87−2.99 (m, 2H), 3.01−3.05 (m, 2H), 3.34 (q, J = 6.3 Hz, 2H), 3.55 (s, 2H), 3.78 (s, 3H), 6.91 (dd, J = 1.3, 8.1 Hz, 1H), 7.20− 7.36 (m, 3H); 13C NMR (101 MHz, CDCl3, APT pulse sequence) δ d (CH, CH3): 10.4, 19.6, 31.1, 43.4, 56.2, 109.9, 124.6, 132.1; u (C, CH2): 24.8, 29.1, 39.7, 41.9, 52.8, 53.6, 58.0, 61.6, 120.2, 125.3, 131.6, 146.6, 155.2, 159.9, 174.9; HRMS calcd for C28H42BrN4O3 [M + H]+ 561.2435; found 561.2434; HPLC purity: 98.4%. N-(3-(cis-3,5-Dimethylpiperidin-1-yl)propyl)-1-((2-(2-chloro-6methoxyphenyl)-5-methyloxazol-4-yl)methyl)piperidine-4-carboxamide 7uu. 1-((2-(2-Chloro-6-methoxyphenyl)-5-methyloxazol-4-yl)methyl)piperidine-4-carboxylic acid (25 mg, 0.069 mmol) and 3-(cis3,5-dimethylpiperidin-1-yl)propan-1-amine (13 mg, 0.075 mmol) were reacted according to general procedure A-3 to afford the product as a viscous, light yellow oil (23 mg, 0.045 mmol, 66% yield). 1H NMR (400 MHz, CDCl3) δ 0.55 (q, J = 11.9 Hz, 1H), 0.87 (d, J = 6.5 Hz, 6H), 1.43 (t, J = 11.1 Hz, 2H), 1.58−1.91 (complex, 9H), 1.97−2.07 (m, 1H), 2.12 (dt, J = 2.8, 11.5 Hz, 2H), 2.40 (s, 3H), 2.43 (t, J = 6.2 Hz, 2H), 2.87−2.91 (m, 2H), 3.01−3.05 (m, 2H), 3.33 (q, J = 6.3 Hz, 2H), 3.53 (s, 2H), 3.80 (s, 3H), 6.87 (dd, J = 0.9, 8.5 Hz, 1H), 7.07 (dd, J = 0.9, 8.1 Hz, 1H), 7.32−7.37 (m, 2H); 13C NMR (101 MHz, CDCl3, APT pulse sequence) δ d (CH, CH3): 10.4, 19.6, 31.2, 43.4, 56.2, 109.4, 121.6, 131.7; u (C, CH2): 24.8, 29.0, 39.8, 42.0, 52.8, 53.6, 58.1, 61.7, 118.0, 135.9, 146.8, 154.1, 159.8, 175.0; HRMS calcd for C28H42ClN4O3 [M + H]+ 517.2940; found 517.2939; HPLC purity: 99.6%. N-(3-(cis-3,5-Dimethylpiperidin-1-yl)propyl)-1-((2-(2-fluoro-6-methoxyphenyl)-5-methyloxazol-4-yl)methyl)piperidine-4-carboxamide 7vv. 1-((2-(2-Fluoro-6-methoxyphenyl)-5-methyloxazol-4-yl)methyl)piperidine-4-carboxylic acid (25 mg, 0.072 mmol) and 3-(cis3,5-dimethylpiperidin-1-yl)propan-1-amine (13 mg, 0.079 mmol) were reacted according to general procedure A-3 to afford the product as a viscous, light yellow oil (21 mg, 0.041 mmol, 58% yield). 1H NMR (400 MHz, CDCl3) δ 0.56 (q, J = 11.9 Hz, 1H), 0.88 (d, J = 6.5 Hz, 6H), 1.44 (t, J = 11.0 Hz, 2H), 1.64−1.91 (complex, 9H), 1.98−2.06 (m, 1H), 2.10 (dt, J = 2.8, 11.4 Hz, 2H), 2.40 (s, 3H), 2.43 (t, J = 6.1 Hz, 2H), 2.86−2.95 (m, 2H), 3.02−3.06 (m, 2H), 3.35 (q, J = 6.3 Hz, 2H), 3.51 (s, 2H), 3.85 (s, 3H), 6.73−6.83 (m, 2H), 7.29−7.42 (m, 2H); 13C NMR (101 MHz, CDCl3, APT pulse sequence) δ d (CH, CH3): 10.5, 19.6, 31.2, 43.4, 56.4, 106.8, 108.2 (d, J = 22.2 Hz), 131.7 (d, J = 10.8 Hz); u (C, CH2): 24.8, 29.0, 39.8, 42.0, 53.1, 53.8, 58.1, 61.7, 132.2, 146.7, 152.1, 159.4 (d, J = 5.8 Hz), 161.7 (d, J = 252.4 Hz), 174.9; HRMS calcd for C28H42FN4O3 [M + H]+ 501.3235; found 501.3228; HPLC purity: >99.8%. N-(3-(cis-3,5-Dimethylpiperidin-1-yl)propyl)-1-((2-(2-chloro-6methoxyphenyl)-5-methyloxazol-4-yl)methyl)piperidine-4-carboxamide 7ww. 1-((2-(2-Methoxy-6-methylphenyl)-5-methyloxazol-4-yl)methyl)piperidine-4-carboxylic acid (25 mg, 0.073 mmol) and 3-(cis3,5-dimethylpiperidin-1-yl)propan-1-amine (14 mg, 0.080 mmol) were reacted according to general procedure A-3 to afford the product as a viscous, light yellow oil (25 mg, 0.050 mmol, 69% yield). 1H NMR (400 MHz, CDCl3) δ 0.55 (q, J = 11.9 Hz, 1H), 0.87 (d, J = 6.5 Hz, 6H), 1.40 (t, J = 11.0 Hz, 2H), 1.58−1.90 (complex, 9H), 1.95−2.05 (m, 1H), 2.10 (dt, J = 2.8, 11.4 Hz, 2H), 2.22 (s, 3H), 2.38 (s, 3H), 2.42 (t, J = 6.1 Hz, 2H), 2.85−2.90 (m, 2H), 3.02−3.06 (m, 2H), 3.34

(q, J = 5.6 Hz, 2H), 3.52 (s, 2H), 3.77 (s, 3H), 6.79 (d, J = 8.3 Hz, 1H), 6.86 (d, J = 7.7 Hz, 1H), 7.24−7.31 (m, 1H), 7.34 (br s, 1H); 13C NMR (101 MHz, CDCl3, APT pulse sequence) δ d (CH, CH3): 10.4, 19.6, 31.4, 41.0, 43.5, 55.9, 108.4, 122.4, 130.7; u (C, CH2): 24.9, 29.1, 40.0, 42.1, 53.0, 53.7, 58.4, 61.8, 118.1, 131.5, 140.2, 146.0, 156.5, 158.7, 174.8; HRMS calcd for C29H45N4O3 [M + H]+ 497.3486; found 497.3483; HPLC purity: >99.8%. N-(3-(cis-3,5-Dimethylpiperidin-1-yl)propyl)-1-((2,6-dimethoxyphenyl)-5-methyloxazol-4-yl)methyl)piperidine-4-carboxamide 7xx. 1-((2-(2,6-Dimethoxyphenyl)-5-methyloxazol-4-yl)methyl)piperidine-4-carboxylic acid (25 mg, 0.069 mmol) and 3-(cis-3,5dimethylpiperidin-1-yl)propan-1-amine (13 mg, 0.076 mmol) were reacted according to general procedure A-3 to afford the product as a viscous, light yellow oil (22 mg, 0.042 mmol, 61% yield). 1H NMR (400 MHz, CDCl3) δ 0.56 (q, J = 12.0 Hz, 1H), 0.88 (d, J = 6.5 Hz, 6H), 1.43 (t, J = 11.0 Hz, 2H), 1.60−1.91 (complex, 9H), 1.97−2.06 (m, 1H), 2.10 (dt, J = 2.7, 11.5 Hz, 2H), 2.39 (s, 3H), 2.43 (t, J = 6.1 Hz, 2H), 2.87−2.91 (m, 2H), 3.03−3.07 (m, 2H), 3.34 (q, J = 6.2 Hz, 2H), 3.51 (s, 2H), 3.78 (s, 6H), 6.59 (d, J = 8.4 Hz, 2H), 7.29 (br s, 1H), 7.35 (t, J = 8.4 Hz, 1H); 13C NMR (101 MHz, CDCl3, APT pulse sequence) δ d (CH, CH3): 10.5, 19.6, 31.3, 43.5, 56.1, 103.8, 131.7; u (C, CH2): 24.9, 29.1, 39.8, 42.0, 53.1, 53.9, 58.2, 61.7, 107.2, 131.7, 146.3, 154.2, 159.7, 175.0; HRMS calcd for C29H45N4O4 [M + H]+ 513.3435; found 513.3431; HPLC purity: >99.8%. N-(3-(cis-3,5-Dimethylpiperidin-1-yl)propyl)-1-((3,4-dimethoxyphenyl)-5-methyloxazol-4-yl)methyl)piperidine-4-carboxamide 7yy. 1-((2-(3,4-Dimethoxyphenyl)-5-methyloxazol-4-yl)methyl)piperidine-4-carboxylic acid (57 mg, 0.16 mmol) and 3-(cis-3,5dimethylpiperidin-1-yl)propan-1-amine (81 mg, 0.47 mmol) were reacted according to general procedure A-1 to afford the product as a white solid (46 mg, 0.090 mmol, 56% yield). 1H NMR (400 MHz, CDCl3) δ 0.56 (q, J = 12.0 Hz, 1H), 0.89 (d, J = 6.5 Hz, 6H), 1.43 (t, J = 11.1 Hz, 2H), 1.60−1.88 (complex, 9H), 2.01−2.12 (m, 3H), 2.40 (s, 3H), 2.43 (t, J = 6.1 Hz, 2H), 2.86−2.93 (m, 2H), 3.00−3.06 (m, 2H), 3.35 (q, J = 5.8 Hz, 2H), 3.45 (s, 2H), 3.94 (s, 3H), 3.98 (s, 3H), 6.93 (d, J = 8.4 Hz, 1H), 7.37 (br s, 1H), 7.55−7.57 (m, 1H), 7.61 (dd, J = 1.8, 8.4 Hz, 1H); 13C NMR (101 MHz, CDCl3, APT pulse sequence) δ d (CH, CH3): 10.3, 19.5, 31.2, 43.3, 55.9, 56.0, 108.9, 110.8, 119.1; u (C, CH2): 24.9, 28.8, 39.7, 42.0, 53.1, 53.9, 58.0, 61.6, 120.7, 132.3, 145.4, 148.9, 150.4, 159.3, 174.8; IR 1502, 1644, 2949 cm−1; HRMS calcd for C29H45N4O4 [M + H]+ 513.3435; found 513.3458; HPLC purity: 94.2%. N-(4-(Diethylamino)butyl)-1-((2-(3,4-dimethoxyphenyl)-5-methyloxazol-4-yl)methyl)piperidine-4-carboxamide 7zz. 1-((2-(3,4-Dimethoxyphenyl)-5-methyloxazol-4-yl)methyl)piperidine-4-carboxylic acid (30 mg, 0.091 mmol) and N1,N1-diethylbutane-1,4-diamine (16 mg, 0.11 mmol) were reacted according to general procedure A-1 to afford the product as a white solid (26 mg, 0.053 mmol, 59% yield). 1 H NMR (400 MHz, CDCl3) δ 1.10 (t, J = 7.2 Hz, 6H), 1.51−1.64 (complex, 4H), 1.77−1.85 (m, 3H), 1.99−2.14 (m, 3H), 2.37 (s, 3H), 2.56 (t, J = 7.2 Hz, 2H), 2.67 (q, J = 7.2 Hz, 4H), 2.98−3.04 (m, 2H), 3.26 (q, J = 5.8 Hz, 2H), 3.42 (s, 2H), 3.59−3.65 (m, 1H), 3.93 (s, 3H), 3.96 (s, 3H), 6.33 (br s, 1H), 6.90 (d, J = 8.4 Hz, 1H), 7.54 (d, J = 1.9 Hz, 1H), 7.58 (dd, J = 2.08.4 Hz, 1H); 13C NMR (101 MHz, CDCl3, APT pulse sequence) δ d (CH, CH3): 8.1, 10.4, 10.6, 43.4, 55.9, 109.0, 110.9, 119.2; u (C, CH2): 23.7, 27.4, 28.8, 38.7, 46.5, 52.1, 53.1, 53.9, 120.8, 132.3, 145.6, 149.0, 150.5, 159.5, 175.1; HRMS calcd for C27H43N4O4 [M + H]+ 487.3279; found 487.3286; HPLC purity: 95.7%. N-(3-(cis-3,5-Dimethylpiperidin-1-yl)propyl)-1-((5-methyl-2(naphthalen-1-yl)oxazol-4-yl)methyl)piperidine-4-carboxamide 7aaa. 1-((5-Methyl-2-(naphthalen-1-yl)oxazol-4-yl)methyl)piperidine-4-carboxylic acid (28 mg, 0.080 mmol) and 3-(cis-3,5dimethylpiperidin-1-yl)propan-1-amine (15 mg, 0.088 mmol) were reacted according to general procedure A-3 to afford the product as a white solid (21 mg, 0.041 mmol, 51% yield). 1H NMR (400 MHz, CDCl3) δ 0.57 (q, J = 11.5 Hz, 1H), 0.88 (d, J = 6.4 Hz, 6H), 1.46 (t, J = 11.0 Hz, 2H), 1.69−1.93 (complex, 9H), 2.01−2.10 (m, 1H), 2.16 (dt, J = 3.0, 11.2 Hz, 2H), 2.46 (t, J = 6.2 Hz, 2H), 2.48 (s, 3H), 2.87− 2.97 (m, 2H), 3.09−3.12 (m, 2H), 3.36 (q, J = 6.4 Hz, 2H), 3.59 (s, 6380

DOI: 10.1021/acs.jmedchem.7b00561 J. Med. Chem. 2017, 60, 6364−6383

Journal of Medicinal Chemistry

Article

6.0 Hz, 2H), 3.48 (s, 2H), 7.03 (t, J = 8.9 Hz, 1H), 7.31 (br s, 1H), 7.52 (s, 1H), 7.77−7.82 (m, 1H), 7.86−7.90 (m, 1H); 13C NMR (101 MHz, CDCl3, APT pulse sequence) δ d (CH, CH3): 14.4 (d, J = 3.5 Hz), 19.6, 31.3, 43.3, 115.4 (d, J = 23.3 Hz), 125.7 (d, J = 8.7 Hz), 129.8 (d, J = 5.7 Hz), 136.1; u (C, CH2): 24.8, 28.9, 39.8, 42.0, 53.2, 54.2, 58.1, 61.7, 123.5 (d, J = 3.6 Hz), 125.5 (d, J = 18.2 Hz), 139.0, 161.0 (d, J = 1.1 Hz), 162.6 (d, J = 250.8 Hz), 174.7; 19F NMR 376 MHz, CDCl3) δ −114.1; IR 1549, 1630, 2944 cm−1; HRMS calcd for C27H40FN4O2 [M + H]+ 471.3130; found 471.3137; HPLC purity: 99.3%. 1-((2-(4-Fluoro-3-methylphenyl)oxazol-4-yl)methyl)-N-(3-(4-isopropylpiperidin-1-yl)propyl)piperidine-4-carboxamide 18b. 1-((2(4-Fluoro-3-methylphenyl)oxazol-4-yl)methyl)piperidine-4-carboxylic acid (40 mg, 0.13 mmol) and 3-(4-isopropylpiperidin-1-yl)propan-1amine (46 mg, 0.25 mmol) were reacted according to general procedure A-3 to afford the product as a viscous light yellow oil (14 mg, 0.028 mmol, 22% yield). 1H NMR (400 MHz, CDCl3) δ 0.80 (d, J = 6.8 Hz, 6H), 0.96−1.06 (m, 1H), 1.24 (dq, J = 2.9, 12.2 Hz, 2H), 1.57−1.84 (complex, 9H), 1.96−2.08 (m, 3H), 2.29 (s, 3H), 2.39 (t, J = 6.1 Hz, 2H), 1.38 (sextet, J = 6.6 Hz, 1H), 1.59−1.68 (complex, 4H), 1.69−1.87 (complex, 5H), 1.97−2.08 (m, 3H), 2.28 (s, 3H), 2.41 (t, J = 6.0 Hz, 2H), 2.94−3.04 (m, 4H), 3.30 (q, J = 5.7 Hz, 2H), 3.47 (s, 2H), 7.02 (t, J = 8.9 Hz, 1H), 7.51 (s, 1H), 7.55 (br s, 1H), 7.75− 7.81 (m, 1H), 7.87 (d, J = 7.1 Hz, 1H); 13C NMR (101 MHz, CDCl3, APT pulse sequence) δ d (CH, CH3): 14.4 (d, J = 3.5 Hz), 19.6, 32.3, 42.1, 43.3, 115.4 (d, J = 23.3 Hz), 125.7 (d, J = 8.7 Hz), 129.8 (d, J = 5.8 Hz), 136.1; u (C, CH2): 24.5, 29.0, 29.1, 39.9, 53.3, 54.2, 54.4, 58.2, 123.5 (d, J = 3.5 Hz), 125.5 (d, J = 18.2 Hz), 138.9, 161.0 (d, J = 1.0 Hz), 162.5 (d, J = 250.7 Hz), 174.8; 19F NMR 376 MHz, CDCl3) δ −114.1; HRMS calcd for C28H42FN4O2 [M + H]+ 485.3286; found 485.3263; HPLC purity: 96.5%. Cells and Viruses. All the cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies, Grand Island, NY, United States) with 10% fetal bovine serum (FBS) (Life Technologies) and antibiotics in 5% CO2 at 37 °C. HCV-Luc was made through insertion of luciferase reporter gene in the HCV JFH-1 strain. Single-round infectious defective HCV particles (HCVsc) and pseudotyped viruses (HCVpp-1a, HCVpp-1b, and VSV-Gpp) were produced as reported before.7,10 HCV-Luc Infection and ATPlite Cytotoxicity Assays. Huh7.5.1 cells seeded in 96-well plates (104 cells/well) were cultured overnight. HCV-Luc virus was used to infect the cells in the presence of titration of the compound of interest. Viral inhibition was evaluated using Renilla Luciferase assay system (Promega, Madison, WI, United States) 48 h post treatment. Cytotoxicity of each compound was measured in parallel using ATPlite assay kit (PerkinElmer, Waltham, MA, United States). EC50 and CC50 values were calculated by nonlinear regression equation in GraphPad Prism 5.0 software (GraphPad Software Inc., La Jolla, CA, United States). HCV Replication Cycle Assays. In HCV single-cycle infection assay, Huh7.5.1 cells seeded in 96-well plates (104 cells/well) were cultured overnight. The cells were inoculated with the infectious HCVsc together with the tested compounds. Luciferase activity of the cells was measured 48 h after the compound treatment. In transient replicon assay, Huh7.5.1 cells seeded in 96-well plates (104 cells/well) were cultured overnight. Then, the cells were transiently transfected with the replicon RNA transcript with DMRIE-C for 4 h. After the transfection reagent was removed, the cells were incubated with DMEM culture medium containing 10 μM of each compound for 48 h. Luciferase activity was measured. In HCV subgenomic replicon assay with HCV replicon (GT 2a) cells, cells were plated into 96-well plate (104 cells/well) and incubated overnight. The cells were treated with tested compounds. Luciferase activity was measured 48 h after the compound treatment. In HCVpp assays, Huh7.5.1 cells were seeded in 96-well plates (104 cells/well) and cultured overnight. Then, the cells were treated with 10 μM of the compounds together with infection of HCVpp GT 1a or VSVpp for 4 h. The cells were then washed and cultured for 48 h followed by a luciferase assay to detect the HCV entry. The results shown are the means of five replicates ± SEM.

2H), 7.34 (br s, 1H), 7.51−7.59 (m, 2H), 7.62−7.66 (m, 1H), 7.89− 7.95 (m, 2H), 8.17 (dd, J = 1.2, 7.3 Hz, 1H), 9.24 (d, J = 8.7 Hz, 1H); 13 C NMR (101 MHz, CDCl3, APT pulse sequence) δ d (CH, CH3): 10.5, 19.6, 31.2, 43.5, 124.9, 126.1, 126.4, 127.3, 127.4, 128.4, 130.6; u (C, CH2): 24.8, 29.1, 39.7, 41.9, 53.1, 54.0, 58.0, 61.6, 124.4, 130.1, 132.8, 133.9, 145.9, 159.2, 174.9; HRMS calcd for C31H43N4O2 [M + H]+ 503.3381; found 503.3386; HPLC purity: >99.8%. N-(3-(cis-3,5-Dimethylpiperidin-1-yl)propyl)-1-((5-methyl-2-(2methylnaphthalen-1-yl)oxazol-4-yl)methyl)piperidine-4-carboxamide 7bbb. 1-((5-Methyl-2-(2-methylnaphthalen-1-yl)oxazol-4-yl)methyl)piperidine-4-carboxylic acid (25 mg, 0.069 mmol) and 3-(cis3,5-dimethylpiperidin-1-yl)propan-1-amine (13 mg, 0.075 mmol) were reacted according to general procedure A-3 to afford the product as a viscous, light yellow oil (20 mg, 0.038 mmol, 55% yield). 1H NMR (400 MHz, CDCl3) δ 0.56 (q, J = 11.8 Hz, 1H), 0.88 (d, J = 6.5 Hz, 6H), 1.44 (t, J = 11.0 Hz, 2H), 1.60−1.95 (complex, 9H), 2.01−2.10 (m, 1H), 2.17 (dt, J = 2.9, 11.4 Hz, 2H), 2.45 (t, J = 6.2 Hz, 2H), 2.461 (s, 3H), 2.464 (s, 3H), 2.88−2.92 (m, 2H), 3.08−3.13 (m, 2H), 3.36 (q, J = 6.3 Hz, 2H), 3.60 (s, 2H), 7.35 (br s, 1H), 7.40 (d, J = 8.4 Hz, 1H), 7.42−7.51 (m, 2H), 7.72−7.75 (m, 1H), 7.79−7.90 (m, 2H); 13C NMR (101 MHz, CDCl3, APT pulse sequence) δ d (CH, CH3): 10.5, 19.6, 20.6, 31.3, 43.4, 125.1, 125.3, 126.9, 127.9, 128.4, 129.9; u (C, CH2): 24.8, 29.1, 39.8, 42.0, 53.1, 53.7, 58.2, 61.7, 124.7, 131.79, 131.83, 132.8, 137.0, 146.3, 158.1, 174.9; HRMS calcd for C32H45N4O2 [M + H]+ 517.3537; found 517.3535; HPLC purity: >99.8%. 1-((2-(Anthracen-9-yl)-5-methyloxazol-4-yl)methyl)-N-(3-(cis-3,5dimethylpiperidin-1-yl)propyl)piperidine-4-carboxamide 7ccc. 1((2-(Anthracen-9-yl)-5-methyloxazol-4-yl)methyl)piperidine-4-carboxylic acid (25 mg, 0.062 mmol) and 3-(cis-3,5-dimethylpiperidin-1yl)propan-1-amine (12 mg, 0.069 mmol) were reacted according to general procedure A-3 to afford the product as a viscous, light yellow oil (16 mg, 0.029 mmol, 47% yield). 1H NMR (400 MHz, CDCl3) δ 0.57 (q, J = 11.2 Hz, 1H), 0.88 (d, J = 6.4 Hz, 6H), 1.51 (t, J = 11.1 Hz, 2H), 1.69−1.98 (complex, 9H), 2.05−2.16 (m, 1H), 2.23 (dt, J = 3.0, 11.1 Hz, 2H), 2.50 (t, J = 6.2 Hz, 2H), 2.53 (s, 3H), 2.91−3.02 (m, 2H), 3.14−3.19 (m, 2H), 3.36 (q, J = 6.4 Hz, 2H), 3.68 (s, 2H), 7.36 (br s, 1H), 7.46−7.58 (complex, 4H), 7.95−8.03 (m, 2H), 8.03− 8.11 (m, 2H), 8.59 (s, 3H); 13C NMR (101 MHz, CDCl3, APT pulse sequence) δ d (CH, CH3): 10.6, 19.5, 30.9, 43.3, 125.4, 125.7, 126.9, 128.5, 129.8; u (C, CH2): 24.7, 29.0, 39.3, 41.7, 53.1, 53.9, 57.6, 61.3, 122.2, 131.1, 131.3, 132.3, 147.0, 157.8, 175.0; HRMS calcd for C35H45N4O2 [M + H]+ 553.3537; found 553.3533; HPLC purity: >99.8%. N-(3-(cis-3,5-Dimethylpiperidin-1-yl)propyl)-1-((5-methyl-2-(otolyl)thiazol-4-yl)methyl)piperidine-4-carboxamide 13. 1-((5-Methyl-2-(o-tolyl)thiazol-4-yl)methyl)piperidine-4-carboxylic acid (56 mg, 0.17 mmol) and 3-(cis-3,5-dimethylpiperidin-1-yl)propan-1-amine (29 mg, 0.17 mmol) were reacted according to general procedure A-1 to afford the product as a white solid (37 mg, 0.077 mmol, 45% yield). 1 H NMR (400 MHz, CDCl3) δ 0.57 (q, J = 12.3 Hz, 1H), 0.87 (d, J = 6.4 Hz, 6H), 1.51 (t, J = 11.0 Hz, 2H), 1.66−1.85 (complex, 9H), 1.98−2.00 (m, 1H), 2.13 (dt, J = 3.2, 11.1 Hz, 2H), 2.47−2.56 (m, 2H), 2.49 (s, 3H), 2.53 (s, 3H), 2.92−2.99 (m, 2H), 2.99−3.06 (m, 2H), 3.32 (q, J = 6.6 Hz, 2H), 3.68 (s, 2H), 7.20−7.30 (complex, 4H), 7.62 (d, J = 7.5 Hz, 1H); 13C NMR (101 MHz, CDCl3, APT pulse sequence) δ d (CH, CH3): 11.4, 19.5, 21.3, 30.9, 43.4, 125.9, 129.0, 129.8, 131.2; u (C, CH2): 24.7, 29.0, 39.2, 41.7, 53.0, 56.0, 57.5, 61.2, 131.1, 133.3, 136.3, 148.9, 163.0, 175.1; IR 1653, 2928, 2960 cm−1; HRMS calcd for C28H43N4OS [M + H]+ 483.3152; found 483.3154; HPLC purity: 97.1%. N-(3-(cis-3,5-Dimethylpiperidin-1-yl)propyl)-1-((2-(4-fluoro-3methylphenyl)oxazol-4-yl)methyl)piperidine-4-carboxamide 18a. 1-((2-(4-Fluoro-3-methylphenyl)oxazol-4-yl)methyl)piperidine-4-carboxylic acid (5.95 g, 18.69 mmol) and 3-(cis-3,5-dimethylpiperidin-1yl)propan-1-amine (3.82 g, 22.43 mmol) were reacted according to general procedure A-3 to afford the product as a white solid (5.30 g, 11.26 mmol, 60% yield). 1H NMR (400 MHz, CDCl3) δ 0.52 (q, J = 12.1 Hz, 1H), 0.84 (d, J = 6.5 Hz, 6H), 1.38 (t, J = 11.1 Hz, 2H), 1.57−1.84 (complex, 9H), 1.96−2.08 (m, 3H), 2.29 (s, 3H), 2.39 (t, J = 6.1 Hz, 2H), 2.82−2.88 (m, 2H), 2.98−3.05 (m, 2H), 3.30 (q, J = 6381

DOI: 10.1021/acs.jmedchem.7b00561 J. Med. Chem. 2017, 60, 6364−6383

Journal of Medicinal Chemistry



Compound Binding Affinity in CNS-Relevant Receptor Panel. Radioligand binding assays using cloned GPCRs, ion channels, and transporters were performed from transiently transfected or stable cell lines as previously detailed21 through the resources of the National Institute of Mental Health Psychoactive Drug Screening Program. Detailed protocols (including cell handling, buffer composition, assay conditions, etc.) for all assays are available online (http://pdspdb.unc. edu/pdspWeb/content/PDSP%20Protocols%20II%202013-03-28. pdf). Initial screening assays were performed using 10 μM (final concentration) of test compound, and the percent inhibition of specific binding by the test compound was determined. When the test compound inhibited >50% of radioligand specific binding, Ki determinations were performed by measuring the inhibition of radioligand binding by various concentrations of test compound (11 concentrations spanning 6 orders of magnitude). Radioligand binding isotherms were regressed using the One Site competition binding function built into GraphPad Prism 4.0 to estimate compound IC50 values. Affinity constants (Ki values) were calculated from IC50 values using the Cheng−Prusoff approximation. In Vitro Combination Test. HCV-Luc infection and ATPlite assays were carried out in 96-well plates in the presences of the compound of interest titrated in vertical and the known antiviral drug titrated in horizontal.13,22 The known antiviral drugs include ribavirin (Sigma-Aldrich), sofosbuvir (Advanced Chemblocks), telaprevir (Selleckchem), daclatasvir (Selleckchem), cyclosporin A (Sigma-Aldrich), and boceprevir (ChemScene). Two independent mathematical models, the Bliss independence model and the Loewe additivity model, were used to predict the theoretical additive, synergistic, or antagonistic effects. By the Bliss independence model, log volumes of synergistic or antagonistic effect were calculated with the MacSynergy program. By the Loewe additivity model, combination indices were calculated at or near the EC50 values of the compound and the antiviral drug when tested alone with CalcuSyn program (Biosoft). Antiviral Screen. The antiviral activity of compound of interest was screened against 13 viruses, including hepatitis B virus, HCV replicon, herpes simplex virus-1, human cytomegalovirus, vaccinia virus, dengue virus, influenza A (H1N1) virus, respiratory syncytial virus, SARS coronavirus, poliovirus 3, Rift Valley fever virus, Tacaribe virus, and Venezuelan equine encephalitis virus, using the nonclinical and preclinical services program offered by the National Institute of Allergy and Infectious Diseases (NIAID) (https://www.niaid.nih.gov/ research/vitro-assessment-antimicrobial-activity-resources-niaid). In Vitro Biopharmaceutical and in Vivo Pharmacokinetic Properties. In vitro biopharmaceutical properties were measured with a fully automated system for sample preparation, sample analysis, and data processing. The microsomal stability of compounds was estimated in rat liver microsomes at 37 °C in the presence of the cofactor NADPH. Concentration of a test article was measured by LC−MS/ MS, and half-life (t1/2) was calculated as described before.23 The solubility of compounds was determined in phosphate buffer (pH 7.4) using μSOL Evolution (Pion Inc., Billerica, MA, United States). The permeability of compounds was estimated via passive diffusion using stirring double-sink parallel artificial membrane permeability assay (PAMPA) (Pion Inc.). The in vivo pharmacokinetics properties of compounds of interest were evaluated in male CD-1 mice after single intraperitoneal administration. Adult male CD-1 mice (25−39 g, n = 3/sampling time point) were obtained from Charles River Laboratories (Wilmington, MA). Mice were housed at the centralized animal facilities at the NIH (Bethesda, MD) with a 12 h light-dark cycle. The housing temperature and relative humidity were controlled at 22 °C and 55%, respectively. The animals had free access to water and food. All experimental procedures were approved by the Animal Care and Use Committee (ACUC) of the NIH Division of Veterinary Resources (DVR). A single dose of 0.1, 1, and 10 mg/kg was administered through i.p. route of administration. The concentration of compounds in the plasma and liver samples was measured by the ultraperformance liquid chromatography−mass spectrometry analysis (UPLC−MS/MS).

Article

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b00561. Tables showing all synthetic intermediates, synthetic procedures, compound characterization for synthetic intermediates, and NIAID antiviral screen data (PDF) Molecular formula strings (SMILES) for final analogues (CSV)



AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Jeffrey Aubé: 0000-0003-1049-5767 Kevin J. Frankowski: 0000-0002-7173-6352 Present Address ⊥

K.J.F, K.L., and J.A.: Center for Integrative Chemical Biology and Drug Discovery, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, 125 Mason Farm Road, Marsico Hall, Chapel Hill, NC 27599-7363, United States.

Notes

The authors declare the following competing financial interest(s): T.J.L., S.H., X.H., Z.H., J.J.M., N.S., J.X., M.F., W.Z., K.J.F., K.L., and F.J.S. are named as inventors on U.S. provisional patent application (no. 62/011,462; Heterocyclic Compounds and Methods of Use Thereof) and international patent application (no. WO 2015192077A1; Preparation of Heterocyclic Compounds Useful in the Treatment of Diseases) related to this work. Other authors declare no competing interests.



ACKNOWLEDGMENTS We thank S. Michael and M. Balcom for assistance in robotic operation in qHTS; P. Shinn, M. Itkin, and D. van Leer for help with compound management; B. Zhang for technical assistance; Benjamin Neuenswander for compound purification and analysis; and Patrick Porubsky for compound management. Receptor binding profile was generously provided by the National Institute of Mental Health’s Psychoactive Drug Screening Program, Contract HHSN-271-2013-00017-C (NIMH PDSP). The NIMH PDSP is directed by Bryan L. Roth MD, Ph.D at the University of North Carolina at Chapel Hill and Project Officer Jamie Driscoll at NIMH, Bethesda, MD, United States. This research was funded by the Intramural Research Program of the National Institute of Diabetes and Digestive and Kidney Diseases, the Intramural Research Program of the National Center for Advancing Translational Sciences, and Molecular Libraries Initiative funding to the University of Kansas Specialized Chemistry Center (U54HG005031).



ABBREVIATIONS USED HCV, hepatitis C virus; IFN-α, interferon α; RBV, ribavirin; DAAs, direct-acting antivirals; HTAs, host-targeting agents; qHTS, high-throughput screening; SAR, structure−activity relationship; DMSO, dimethyl sulfoxide; HCV-Luc, HCV JFH-1 strain with insertion of the luciferase reporter gene; 6382

DOI: 10.1021/acs.jmedchem.7b00561 J. Med. Chem. 2017, 60, 6364−6383

Journal of Medicinal Chemistry

Article

chlorcyclizine derivatives for the treatment of hepatitis C virus infection. J. Med. Chem. 2016, 59, 841−853. (17) Schweitzer, C. J.; Liang, T. J. Border control in hepatitis C virus infection: inhibiting viral entry. ACS Infect. Dis. 2015, 1, 416−419. (18) Wang, H.; Wang, S.; Cheng, L.; Chen, L.; Wang, Y.; Qing, J.; Huang, S.; Wang, Y.; Lei, X.; Wu, Y.; Ma, Z. Discovery of imidazo [1, 2-α][1, 8] naphthyridine derivatives as potential HCV entry inhibitor. ACS Med. Chem. Lett. 2015, 6, 977−981. (19) Mittapalli, G. K.; Jackson, A.; Zhao, F.; Lee, H.; Chow, S.; McKelvy, J.; Wong-Staal, F.; Macdonald, J. E. Discovery of highly potent small molecule hepatitis C virus entry inhibitors. Bioorg. Med. Chem. Lett. 2011, 21, 6852−6855. (20) Baldick, C. J.; Wichroski, M. J.; Pendri, A.; Walsh, A. W.; Fang, J.; Mazzucco, C. E.; Pokornowski, K. A.; Rose, R. E.; Eggers, B. J.; Hsu, M.; Zhai, W. A novel small molecule inhibitor of hepatitis C virus entry. PLoS Pathog. 2010, 6, e1001086. (21) Besnard, J.; Ruda, G. F.; Setola, V.; Abecassis, K.; Rodriguiz, R. M.; Huang, X. P.; Norval, S.; Sassano, M. F.; Shin, A. I.; Webster, L. A.; Simeons, F. R.; Stojanovski, L.; Prat, A.; Seidah, N. G.; Constam, D. B.; Bickerton, G. R.; Read, K. D.; Wetsel, W. C.; Gilbert, I. H.; Roth, B. L.; Hopkins, A. L. Automated design of ligands to polypharmacological profiles. Nature 2012, 492, 215−220. (22) Lin, B.; He, S.; Yim, H. J.; Liang, T. J.; Hu, Z. Evaluation of antiviral drug synergy in an infectious HCV system. Antiviral Ther. 2016, 21, 595−603. (23) Obach, R. S. Prediction of human clearance of twenty-nine drugs from hepatic microsomal intrinsic clearance data: An examination of in vitro half-life approach and nonspecific binding to microsomes. Drug Metab. Dispos. 1999, 27, 1350−1359.

EC50, the concentration of compound that inhibited 50% of virus level of DMSO; CC50, the concentration of compound that exhibited 50% of cytotoxicity of DMSO; HCVsc, singleround infectious defective HCV particles; HCVpp, HCV pseudoparticle; RLU, relative luminescence units; ADME, absorption, distribution, metabolism, and excretion; i.p, intraperitoneal; DMF, N,N-dimethylformamide; LC−MS, liquid chromatography−mass spectrometry; TOF, time-of-flight; TFA, trifluoroacetic acid; NCS, N-chlorosuccinimide; SFC, supercritical fluid chromatography; THF, tetrahydrofuran; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; NIAID, National Institute of Allergy and Infectious Diseases



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DOI: 10.1021/acs.jmedchem.7b00561 J. Med. Chem. 2017, 60, 6364−6383